Compositions for modulating SOD-1 expression

ABSTRACT

Disclosed herein are antisense compounds and methods for decreasing SOD-1 mRNA and protein expression. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate SOD-1 associated diseases, disorders, and conditions. Such SOD-1 associated diseases include amyotrophic sclerosis (ALS).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priority of U.S. application Ser. No. 16/513,297, filed Jul. 16, 2019 (now U.S. Pat. No. 10,669,546), which is a divisional of U.S. application Ser. No. 15/301,004 filed Sep. 30, 2016 (now U.S. Pat. No. 10,385,341), which is U.S. national stage application of PCT/US2015/023934, filed Apr. 1, 2015, which in turn claims the benefit of priority of U.S. Provisional Appl. No. 61/973,803 filed Apr. 1, 2014. The contents of each of these applications are incorporated by reference herein in their entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0240WOSEQ_ST25.pdf created Mar. 30, 2015, which is 320 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

Provided are compositions and methods for reducing expression of superoxide dismutase 1, soluble (SOD-1) mRNA and protein in an animal. Such methods are useful to treat, prevent, or ameliorate neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) by inhibiting expression of SOD-1 in an animal.

BACKGROUND

The soluble SOD-1 enzyme (also known as Cu/Zn superoxide dismutase) is one of the superoxide dismutases that provide defense against oxidative damage of biomolecules by catalyzing the dismutation of superoxide to hydrogen peroxide (H₂O₂) (Fridovich, Annu. Rev. Biochem., 1995, 64, 97-112). The superoxide anion (02-) is a potentially harmful cellular by-product produced primarily by errors of oxidative phosphorylation in mitochondria (Turrens, J. Physiol. 2003, 552, 335-344) Mutations in the SOD-1 gene are associated with a dominantly-inherited form of amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) a disorder characterized by a selective degeneration of upper and lower motor neurons (Rowland, N. Engl. J. Med. 2001, 344, 1688-1700). There is a tight genetic linkage between familial ALS and missense mutations in the SOD1 gene (Rosen, Nature, 1993, 362, 59-62). The toxicity of mutant SOD1 is believed to arise from an initial misfolding (gain of function) reducing nuclear protection from the active enzyme (loss of function in the nuclei), a process that may be involved in ALS pathogenesis (Sau, Hum. Mol. Genet. 2007, 16, 1604-1618).

ALS is a devastating progressive neurodegenerative disease affecting as many as 30,000 Americans at any given time. The progressive degeneration of the motor neurons in ALS eventually leads to their death. When the motor neurons die, the ability of the brain to initiate and control muscle movement is lost. With voluntary muscle action progressively affected, patients in the later stages of the disease may become totally paralyzed.

Currently lacking are acceptable options for treating such neurodegenerative diseases. It is therefore an object herein to provide methods for the treatment of such diseases.

SUMMARY

Provided herein are methods, compounds, and compositions for modulating expression of superoxide dismutase 1, soluble (SOD-1) mRNA and protein. In certain embodiments, compounds useful for modulating expression of SOD-1 mRNA and protein are antisense compounds. In certain embodiments, the antisense compounds are modified oligonucleotides.

In certain embodiments, modulation can occur in a cell or tissue. In certain embodiments, the cell or tissue is in an animal. In certain embodiments, the animal is a human. In certain embodiments, SOD-1 mRNA levels are reduced. In certain embodiments, SOD-1 protein levels are reduced. Such reduction can occur in a time-dependent manner or in a dose-dependent manner.

Also provided are methods, compounds, and compositions useful for preventing, treating, and ameliorating diseases, disorders, and conditions. In certain embodiments, such SOD-1 related diseases, disorders, and conditions are neurodegenerative diseases. In certain embodiments, such neurodegenerative diseases, disorders, and conditions include amyotrophic lateral sclerosis (ALS).

Such diseases, disorders, and conditions can have one or more risk factors, causes, or outcomes in common. Certain risk factors and causes for development of ALS include growing older, having a personal or family history, or genetic predisposition. However, the majority of ALS cases are sporadic and no known risk factors are known. Certain symptoms and outcomes associated with development of ALS include but are not limited to: fasciculations, cramps, tight and stiff muscles (spasticity), muscle weakness affecting an arm or a leg, slurred and nasal speech, difficulty walking, difficulty chewing or swallowing (dysphagia), difficulty speaking or forming words (dysarthria), weakness or atrophy, spasticity, exaggerated reflexes (hyperreflexia), and presence of Babinski's sign. As ALS progresses, symptoms and outcomes by include weakening of other limbs, perhaps accompanied by twitching, muscle cramping, and exaggerated, faster reflexes; problems with chewing, swallowing, and breathing; drooling may occur; eventual paralysis; and death.

In certain embodiments, methods of treatment include administering an SOD-1 antisense compound to an individual in need thereof. In certain embodiments, methods of treatment include administering an SOD-1 modified oligonucleotide to an individual in need thereof.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Additionally, as used herein, the use of “and” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise. Also, all sequences described herein are listed 5′ to 3′, unless otherwise stated. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this disclosure, including, but not limited to, patents, patent applications, published patent applications, articles, books, treatises, and GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis.

Unless otherwise indicated, the following terms have the following meanings:

“2′-deoxynucleoside” (also 2′-deoxyribonucleoside) means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).

“2′-deoxyribose sugar” means a 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA).

“2′-O-methoxyethyl” (also 2′-MOE and 2′-OCH₂CH₂—OCH₃ and MOE and 2′-O-methoxyethylribose) refers to an O-methoxy-ethyl modification of the 2′ position of a furanose ring. A 2′-O-methoxyethylribose modified sugar is a modified sugar.

“2′-O-methoxyethylribose modified nucleoside” (also 2′-MOE nucleoside) means a nucleoside comprising a 2′-MOE modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position of the furanose ring other than H or OH. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications.

“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.

“About” means within 10% of a value. For example, if it is stated, “the compounds affected at least about 50% inhibition of SOD-1”, it is implied that the SOD-1 levels are inhibited within a range of 45% and 55%. “Administered concomitantly” refers to the co-administration of two pharmaceutical agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both pharmaceutical agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both pharmaceutical agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.

“Administering” means providing a pharmaceutical agent to an animal, and includes, but is not limited to administering by a medical professional and self-administering.

“Amelioration” refers to a lessening, slowing, stopping, or reversing of at least one indicator of the seventy of a condition or disease. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

“Antibody” refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, Fab region, and Fc region.

“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or in the absence of the antisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridization of a compound with a target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.

“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding segment of a target nucleic acid.

“Base complementarity” refers to the capacity for the precise base pairing of nucleobases of an oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.

“Bicyclic sugar” means a furanose ring modified by the bridging of two atoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotide wherein the furanose portion of the nucleoside or nucleotide includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.

“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.

“cEt” or “constrained ethyl” or “cEt modified sugar” means a bicyclic nucleoside having a sugar moiety comprising a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH₃)—O-2′. A cEt modified sugar is a modified sugar.

“cEt modified nucleoside” means a bicyclic nucleoside having a sugar moiety comprising a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH₃)—O-2′. A cEt modified sugar is a modified sugar.

“Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′-O-methoxyethyl nucleosides is chemically distinct from a region having nucleosides without 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has at least two chemically distinct regions, each position having a plurality of subunits.

“Co-administration” means administration of two or more pharmaceutical agents to an individual.

The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.

“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

“Comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

“Contiguous nucleobases” means nucleobases immediately adjacent to each other.

“Designing” or “designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.

“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, in drugs that are injected, the diluent may be a liquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month.

“Effective amount” in the context of modulating an activity or of treating or preventing a condition means the administration of that amount of pharmaceutical agent to a subject in need of such modulation, treatment, or prophylaxis, either in a single dose or as part of a series, that is effective for modulation of that effect, or for treatment or prophylaxis or improvement of that condition. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.

“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as a “gap” and the external regions may be referred to as the “wings.”

“Gap-narrowed” means a chimeric antisense compound having a gap segment of 9 or fewer contiguous 2′-deoxyribonucleosides positioned between and immediately adjacent to 5′ and 3′ wing segments having from 1 to 6 nucleosides.

“Gap-widened” means a chimeric antisense compound having a gap segment of 12 or more contiguous 2′-deoxyribonucleosides positioned between and immediately adjacent to 5′ and 3′ wing segments having from 1 to 6 nucleosides.

“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a target nucleic acid. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an oligonucleotide and a nucleic acid target.

“Identifying an animal having a SOD-1 associated disease” means identifying an animal having been diagnosed with a SOD-1 associated disease or predisposed to develop a SOD-1 associated disease. Individuals predisposed to develop a SOD-1 associated disease include those having one or more risk factors for developing a SOD-1 associated disease, including, growing older, having a personal or family history, or genetic predisposition of one or more SOD-1 associated diseases. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments, such as genetic testing.

“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment or therapy.

“Inhibiting SOD-1” means reducing the level or expression of a SOD-1 mRNA and/or protein. In certain embodiments, SOD-1 mRNA and/or protein levels are inhibited in the presence of an antisense compound targeting SOD-1, including a modified oligonucleotide targeting SOD-1, as compared to expression of SOD-1 mRNA and/or protein levels in the absence of a SOD-1 antisense compound, such as a modified oligonucleotide.

“Inhibiting the expression or activity” refers to a reduction or blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.

“Internucleoside linkage” refers to the chemical bond between nucleosides.

“Linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.

“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.

“Mixed backbone” means a pattern of internucleoside linkages including at least two different internucleoside linkages. For example, an oligonucleotide with a mixed backbone may include at least one phosphodiester linkage and at least one phosphorothioate linkage.

“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e., a phosphodiester internucleoside bond).

“Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

A “modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, and/or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, modified sugar, and/or modified nucleobase.

“Modified sugar” means substitution and/or any change from a natural sugar moiety.

“Monomer” means a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

“Motif” means the pattern of unmodified and modified nucleosides in an antisense compound.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH).

“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

“Non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.

“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo, or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.

“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

“Off-target effect” refers to an unwanted or deleterious biological effect associated with modulation of RNA or protein expression of a gene other than the intended target nucleic acid.

“Oligomeric compound” or “oligomer” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.

“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection (e.g., bolus injection) or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g., intrathecal or intracerebroventricular administration.

“Peptide” means a molecule formed by linking at least two amino acids by amide bonds. Without limitation, as used herein, peptide refers to polypeptides and proteins.

“Pharmaceutical agent” means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, a modified oligonucleotide targeted to SOD-1 is a pharmaceutical agent.

“Pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise a modified oligonucleotide and a sterile aqueous solution.

“Pharmaceutically acceptable derivative” encompasses pharmaceutically acceptable salts, conjugates, prodrugs or isomers of the compounds described herein.

“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.

“Prevent” or “preventing” refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to days, weeks to months, or indefinitely.

“Prodrug” means a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.

“Prophylactically effective amount” refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.

“Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.

“Salts” mean a physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

“Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.

“Shortened” or “truncated” versions of oligonucleotides SOD-1ght herein have one, two or more nucleosides deleted.

“Side effects” means physiological responses attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies.

“Single-stranded oligonucleotide” means an oligonucleotide which is not hybridized to a complementary strand.

“Sites,” as used herein, are defined as unique nucleobase positions within a target nucleic acid.

“Slows progression” means decrease in the development of the disease.

“SOD-1” means the mammalian gene superoxide dismutase 1, soluble (SOD-1), including the human gene superoxide dismutase 1, soluble (SOD-1).

“SOD-1 associated disease” means any disease associated with any SOD-1 nucleic acid or expression product thereof. Such diseases may include a neurodegenerative disease. Such neurodegenerative diseases may include amyotrophic lateral sclerosis (ALS).

“SOD-1 mRNA” means any messenger RNA expression product of a DNA sequence encoding SOD-1.

“SOD-1 nucleic acid” means any nucleic acid encoding SOD-1. For example, in certain embodiments, a SOD-1 nucleic acid includes a DNA sequence encoding SOD-1, an RNA sequence transcribed from DNA encoding SOD-1 (including genomic DNA comprising introns and exons), and a mRNA sequence encoding SOD-1. “SOD-1 mRNA” means a mRNA encoding a SOD-1 protein.

“SOD-1 protein” means the polypeptide expression product of a SOD-1 nucleic acid.

“Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments.

“Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.

“Subject” means a human or non-human animal selected for treatment or therapy.

“Sugar chemistry motif” means a pattern of sugar modifications including at least two different sugar modifications. For example, an oligonucleotide with a mixed backbone may include at least one 2′-O-methoxyethyl modified nucleoside, and/or one cEt modified nucleoside, and/or one 2′-deoxynucleoside.

“Target” refers to a protein, the modulation of which is desired.

“Target gene” refers to a gene encoding a target.

“Targeting” or “targeted” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by antisense compounds.

“Target region” means a portion of a target nucleic acid to which one or more antisense compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.

“Treat” or “treating” or “treatment” refers administering a composition to effect an alteration or improvement of the disease or condition.

“Unmodified nucleobases” mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Unmodified nucleotide” means a nucleotide composed of naturally occuring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

“Wing segment” means a plurality of nucleosides modified to impart to an oligonucleotide properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Certain Embodiments

Certain embodiments provide methods, compounds, and compositions for inhibiting SOD-1 mRNA and protein expression. Certain embodiments provide methods, compounds, and composition for decreasing SOD-1 mRNA and protein levels.

Certain embodiments provide antisense compounds targeted to a SOD-1 nucleic acid. In certain embodiments, the SOD-1 nucleic acid is the sequence set forth in GENBANK Accession No. NM_000454.4 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000 to 18704000 (incorporated herein as SEQ ID NO: 2), and the complement of GENBANK Accession No. NW_001114168.1 truncated from nucleotides 2258000 to U.S. Pat. No. 2,271,000 (incorporated herein as SEQ ID NO: 3).

Certain embodiments provide methods for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with SOD-1 in an individual in need thereof. Also contemplated are methods for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with SOD-1. SOD-1 associated diseases, disorders, and conditions include neurodegenerative diseases. In certain embodiments, SOD-1 associated diseases include amyotrophic lateral sclerosis (ALS).

Embodiment 1

-   -   A compound, comprising a modified oligonucleotide consisting of         12 to 30 linked nucleosides and having a nucleobase sequence         comprising at least 8, at least 9, at least 10, at least 11, at         least 12, at least 13, at least 14, at least 15, at least 16, at         least 17, at least 18, at least 19, or at least 20 consecutive         nucleobases of any of the nucleobase sequences of SEQ ID NOs:         118-1461.

Embodiment 2

-   -   A compound, comprising a modified oligonucleotide consisting of         12 to 30 linked nucleosides and having a nucleobase sequence         comprising at least 8, at least 9, at least 10, at least 11, at         least 12, at least 13, at least 14, at least 15, at least 16, at         least 17, at least 18, at least 19, or at least 20 consecutive         nucleobases of any of the nucleobase sequences of SEQ ID NOs:15,         21, 23, 47, 54, and 67, wherein at least one internucleoside         linkage is a phosphodiester linkage.

Embodiment 3

-   -   The compound of any preceding embodiment, wherein the modified         oligonucleotide has a mixed backbone.

Embodiment 4

-   -   The compound of embodiment 3, wherein the mixed backbone motif         is any of the following:         -   sossssssssoooss,         -   sooossssssssoss,         -   sooosssssssssoss,         -   soosssssssssooss,         -   sooossssssssooss,         -   sooosssssssssooss,         -   sooossssssssssooss,         -   sooosssssssssssooos,         -   soooossssssssssooss,         -   sooosssssssssssooss,         -   sososssssssssssosos, and         -   sooossssssssssoooss, wherein         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 5

-   -   The compound of any preceding embodiment, wherein the modified         oligonucleotide has a sugar chemistry motif of any of the         following:         -   ekddddddddekekee,         -   kekeddddddddekek,         -   eeeedddddddddkkee,         -   eeeeddddddddekeke,         -   eeeeddddddddkekee,         -   eeeeddddddddkkeee,         -   eeeeeddddddddkkee,         -   eeeekddddddddkeee,         -   eeeekdddddddkeeee,         -   eeekddddddddkeeee,         -   eeekkdddddddkkeee,         -   eekkdddddddddkkee,         -   eekkddddddddeeeee,         -   eekkddddddddkkeee,         -   ekekddddddddeeeee,         -   ekekddddddddkekee, and         -   kekeddddddddeeeee, wherein         -   e=a 2′-O-methoxyethylribose modified sugar,         -   k=a cEt modified sugar,         -   d=a 2′-deoxyribose sugar,

Embodiment 6

-   -   The compound of any preceding embodiment, wherein the nucleobase         sequence of the modified oligonucleotide is at least 80%, at         least 81%, at least 82%, at least 83%, at least 84%, at least         85%, at least 86%, at least 87%, at least 88%, at least 89%, at         least 90%, at least 91%, at least 92%, at least 93%, at least         94%, at least 95%, at least 96%, at least 97%, at least 98%, at         least 99%, or 100% complementary to SEQ ID NO: 1 or SEQ ID NO:         2.

Embodiment 7

-   -   The compound of any preceding embodiment, consisting of a         single-stranded modified oligonucleotide.

Embodiment 8

-   -   The compound of any preceding embodiment, wherein at least one         internucleoside linkage is a modified internucleoside linkage.

Embodiment 9

-   -   The compound of embodiment 8, wherein at least one modified         internucleoside linkage is a phosphorothioate internucleoside         linkage.

Embodiment 10

-   -   The compound of embodiment 9, wherein each modified         internucleoside linkage is a phosphorothioate internucleoside         linkage.

Embodiment 11

-   -   The compound of any preceding embodiment, wherein at least one         internucleoside linkage is a phosphodiester internucleoside         linkage.

Embodiment 12

-   -   The compound of any preceding embodiment, wherein at least one         internucleoside linkage is a phosphorothioate linkage and at         least one internucleoside linkage is a phosphodiester linkage.

Embodiment 13

-   -   The compound of any preceding embodiment, wherein at least one         nucleoside comprises a modified nucleobase.

Embodiment 14

-   -   The compound of embodiment 13, wherein the modified nucleobase         is a 5-methylcytosine.

Embodiment 15

-   -   The compound of any preceding embodiment, wherein at least one         nucleoside of the modified oligonucleotide comprises a modified         sugar.

Embodiment 16

-   -   The compound of embodiment 15, wherein the at least one modified         sugar is a bicyclic sugar.

Embodiment 17

-   -   The compound of embodiment 16, wherein the bicyclic sugar         comprises a chemical link between the 2′ and 4′ position of the         sugar 4′-CH₂—N(R)—O-2′ bridge wherein R is, independently, H,         C₁-C₁₂ alkyl, or a protecting group.

Embodiment 18

-   -   The compound of embodiment 17, wherein the bicyclic sugar         comprises a 4′-CH₂—N(R)—O-2′ bridge wherein R is, independently,         H, C₁-C₁₂ alkyl, or a protecting group.

Embodiment 19

-   -   The compound of embodiment 15, wherein at least one modified         sugar comprises a 2′-O-methoxyethyl group.

Embodiment 20

-   -   The compound of embodiment 15, wherein the modified sugar         comprises a 2′-O(CH₂)₂—OCH₃ group.

Embodiment 21

-   -   The compound of any preceding embodiment, wherein the modified         oligonucleotide comprises:         -   a gap segment consisting of 10 linked deoxynucleosides;         -   a 5′ wing segment consisting of 5 linked nucleosides; and         -   a 3′ wing segment consisting of 5 linked nucleosides;         -   wherein the gap segment is positioned between the 5′ wing             segment and the 3′ wing segment and wherein each nucleoside             of each wing segment comprises a modified sugar.

Embodiment 22

-   -   The compound of any preceding embodiment, wherein the modified         oligonucleotide comprises:         -   a gap segment consisting of 9 linked deoxynucleosides;         -   a 5′ wing segment consisting of 5 linked nucleosides; and         -   a 3′ wing segment consisting of 5 linked nucleosides;         -   wherein the gap segment is positioned between the 5′ wing             segment and the 3′ wing segment and wherein each nucleoside             of each wing segment comprises a modified sugar.

Embodiment 23

-   -   The compound of any preceding embodiment, wherein the modified         oligonucleotide comprises:         -   a gap segment consisting of 8 linked deoxynucleosides;         -   a 5′ wing segment consisting of 5 linked nucleosides; and         -   a 3′ wing segment consisting of 5 linked nucleosides;         -   wherein the gap segment is positioned between the 5′ wing             segment and the 3′ wing segment and wherein each nucleoside             of each wing segment comprises a modified sugar.

Embodiment 24

-   -   The compound of any preceding embodiment, wherein the modified         oligonucleotide comprises:         -   a gap segment consisting of 8 linked deoxynucleosides;         -   a 5′ wing segment consisting of 4 linked nucleosides; and         -   a 3′ wing segment consisting of 5 linked nucleosides;         -   wherein the gap segment is positioned between the 5′ wing             segment and the 3′ wing segment and wherein each nucleoside             of each wing segment comprises a modified sugar.

Embodiment 25

-   -   The compound of any preceding embodiment, wherein the modified         oligonucleotide comprises:         -   a gap segment consisting of 8 linked deoxynucleosides;         -   a 5′ wing segment consisting of 5 linked nucleosides; and         -   a 3′ wing segment consisting of 7 linked nucleosides;         -   wherein the gap segment is positioned between the 5′ wing             segment and the 3′ wing segment and wherein each nucleoside             of each wing segment comprises a modified sugar.

Embodiment 26

-   -   The compound of any preceding embodiment, wherein the modified         oligonucleotide comprises:         -   a gap segment consisting of 8 linked deoxynucleosides;         -   a 5′ wing segment consisting of 6 linked nucleosides; and         -   a 3′ wing segment consisting of 6 linked nucleosides;         -   wherein the gap segment is positioned between the 5′ wing             segment and the 3′ wing segment and wherein each nucleoside             of each wing segment comprises a modified sugar.

Embodiment 27

-   -   The compound of any preceding embodiment, wherein the modified         oligonucleotide comprises:         -   a gap segment consisting of 9 linked deoxynucleosides;         -   a 5′ wing segment consisting of 6 linked nucleosides; and         -   a 3′ wing segment consisting of 5 linked nucleosides;         -   wherein the gap segment is positioned between the 5′ wing             segment and the 3′ wing segment and wherein each nucleoside             of each wing segment comprises a modified sugar.

Embodiment 28

-   -   The compound of any preceding embodiment, wherein the modified         oligonucleotide consists of 12, 13, 14, 15, 16, 17, 18, 19, or         20 linked nucleosides.

Embodiment 29

-   -   A compound consisting of a modified oligonucleotide according to         the following formula:

Embodiment 30

-   -   A compound consisting of a modified oligonucleotide according to         the following formula:

Embodiment 31

-   -   A compound consisting of a modified oligonucleotide according to         the following formula:

Embodiment 32

-   -   A compound consisting of a modified oligonucleotide according to         the following formula:

Embodiment 33

-   -   A compound consisting of a modified oligonucleotide according to         the following formula:

Embodiment 34

-   -   A compound consisting of a modified oligonucleotide according to         the following formula:

Embodiment 35

-   -   A compound consisting of a modified oligonucleotide according to         the following formula:

Embodiment 36

-   -   A compound consisting of a modified oligonucleotide according to         the following formula:

Embodiment 37

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds         Tds Tds mCds Tds Ads mCeo Aes Geo mCes Te; wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 38

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Tes Teo Aeo Aes Tds Gds Tds Tds Tds Ads         Tds mCds Ako Gko Ges Aes Te; wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   k=a cEt modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 39

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Ges Geo Aeo Teo Ads mCds Ads Tds Tds Tds         mCds Tds Ads mCko Aks Ges mCe; wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   k=a cEt modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 40

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Ges Geo Aeo Teo Aes mCds Ads Tds Tds Tds         mCds Tds Ads mCko Aks Ges mCe; wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   k=a cEt modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 41

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Ges Geo Aeo Teo Aks mCds Ads Tds Tds Tds         mCds Tds Ads mCko Aes Ges mCe; wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   k=a cEt modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 42

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Aes Gko Teo Gks Tds Tds Tds Ads Ads Tds         Gds Tds Tko Teo Aks Tes mCe; wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   k=a cEt modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 43

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Aes Gko Teo Gks Tds Tds Tds Ads Ads Tds         Gds Tds Teo Teo Aes Tes mCe; wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   k=a cEt modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 44

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Aes Geo Tko Gks Tds Tds Tds Ads Ads Tds         Gds Tds Teo Teo Aes Tes mCe; wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   k=a cEt modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 45

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: mCes mCeo Geo Teo mCeo Gds mCds mCds mCds         Tds Tds mCds Ads Gds mCds Aeo mCeo Ges mCes Ae, wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 46

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: mCes mCeo Geo Teo mCes Gds mCds mCds mCds         Tds Tds mCds Ads Ges mCeo Aeo mCeo Ges mCes Ae, wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 47

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: mCes mCeo Geo Teo mCes Gds mCds mCds mCds         Tds Tds mCds Ads Gds mCds Aeo mCeo Geo mCes Ae, wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 48

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Aes mCeo Aeo mCeo mCes Tds Tds mCds Ads         mCds Tds Gds Gds Tds mCds mCeo Aeo Teo Tes Ae, wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 49

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Ges Geo mCeo Geo Aes Tds mCds mCds mCds         Ads Ads Tds Tds Ads mCds Aeo mCeo mCeo Aes mCe, wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 50

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Ges Geo mCeo Geo Aes Tes mCds mCds mCds         Ads Ads Tds Tds Ads mCeo Aeo mCeo mCes Aes mCe, wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 51

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Ges Geo mCeo Geo Aes Tds mCds mCds mCds         Ads Ads Tds Tds Aes mCeo Aeo mCeo mCes Aes mCe, wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 52

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Ges Geo mCeo Geo Aeo Tes mCds mCds mCds         Ads Ads Tds Tds Ads mCds Aeo mCeo mCes Aes mCe, wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 53

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Ges Teo mCeo Geo mCes mCds mCds Tds Tds         mCds Ads Gds mCds Ads mCds Geo mCeo Aeo mCes Ae, wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 54

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Tes mCeo Geo mCeo mCes mCds Tds Tds mCds         Ads Gds mCds Ads mCds Gds mCeo Aeo mCeo Aes mCe, wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar,         -   s=a phosphorothioate internucleoside linkage, and         -   o=a phosphodiester internucleoside linkage.

Embodiment 55

-   -   A compound consisting of a modified oligonucleotide according to         the following formula: Ges Aes Aes Aes Tes Tds Gds Ads Tds Gds         Ads Tds Gds mCds mCds mCes Tes Ges mCes Ae, wherein,         -   A=an adenine,         -   mC=a 5′-methylcytosine         -   G=a guanine,         -   T=a thymine,         -   e=a 2′-O-methoxyethylribose modified sugar,         -   d=a 2′-deoxyribose sugar, and         -   s=a phosphorothioate internucleoside linkage.

Embodiment 56

-   -   A composition comprising the compound of any preceding         embodiment or salt thereof and at least one of a         pharmaceutically acceptable carrier or diluent.

Embodiment 57

-   -   A method comprising administering to an animal the compound or         composition of any preceding embodiment.

Embodiment 58

-   -   The method of embodiment 57, wherein the animal is a human.

Embodiment 59

-   -   The method of embodiment 57, wherein administering the compound         prevents, treats, ameliorates, or slows progression of a SOD-1         associated disease.

Embodiment 60

-   -   The method of embodiment 59, wherein the SOD-1 associated         disease is a neurodegenerative disease.

Embodiment 61

-   -   The method of embodiment 60, wherein the SOD-1 associated         disease is ALS.

Embodiment 62

-   -   Use of the compound or composition of any preceding embodiment         for the manufacture of a medicament for treating a         neurodegenerative disorder.

Embodiment 63

-   -   Use of the compound or composition of any preceding embodiment         for the manufacture of a medicament for treating ALS.

Embodiment 64

-   -   The compound or composition of any preceding embodiment wherein         the modified oligonucleotide does not have the nucleobase         sequence of SEQ ID NO: 21.

Embodiment 65

-   -   The compound or composition of any preceding embodiment wherein         the modified oligonucleotide does not have the nucleobase         sequence of any of SEQ ID NOs: 21-118.

Embodiment 66

-   -   A compound comprising a modified oligonucleotide consisting of         12 to 30 linked nucleosides and having a nucleobase sequence,         wherein the nucleobase sequence comprises an at least 12         consecutive nucleobase portion complementary to an equal number         of nucleobases of nucleotides 665 to 684 of SEQ ID NO: 1,         wherein the modified oligonucleotide is at least 80%         complementary to SEQ ID NO: 1.

Embodiment 67

-   -   The compound of embodiment 66, wherein the modified         oligonucleotide is 100% complementary to SEQ ID NO: 1.

Embodiment 68

-   -   The compound of embodiment 66, wherein the modified         oligonucleotide is a single-stranded modified oligonucleotide.

Embodiment 69

-   -   The compound of embodiments 66-68 wherein at least one         internucleoside linkage is a modified internucleoside linkage.

Embodiment 70

-   -   The compound of embodiment 69, wherein at least one modified         internucleoside linkage is a phosphorothioate internucleoside         linkage.

Embodiment 71

-   -   The compound of embodiment 70, wherein each modified         internucleoside linkage is a phosphorothioate internucleoside         linkage.

Embodiment 72

-   -   The compound of embodiments 66-69, wherein at least one         internucleoside linkage is a phosphodiester internucleoside         linkage.

Embodiment 73

-   -   The compound of embodiments 66-71 and 72-73, wherein at least         one internucleoside linkage is a phosphorothioate linkage and at         least one internucleoside linkage is a phosphodiester linkage.

Embodiment 74

-   -   The compound of embodiments 66-73, wherein at least one         nucleoside comprises a modified nucleobase.

Embodiment 75

-   -   The compound of embodiment 74, wherein the modified nucleobase         is a 5-methylcytosine.

Embodiment 76

-   -   The compound of embodiments 66-75, wherein at least one         nucleoside of the modified oligonucleotide comprises a modified         sugar.

Embodiment 77

-   -   The compound of embodiment 76, wherein the at least one modified         sugar is a bicyclic sugar.

Embodiment 78

-   -   The compound of embodiment 77, wherein the bicyclic sugar         comprises a 4′-CH(R)—O-2′ bridge wherein R is, independently, H,         C₁-C₂ alkyl, or a protecting group.

Embodiment 79

-   -   The compound of embodiment 78, wherein R is methyl.

Embodiment 80

-   -   The compound of embodiment 78, wherein R is H.

Embodiment 81

-   -   The compound of embodiment 76, wherein the at least one modified         sugar comprises a 2′-O-methoxyethyl group.         Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, modified oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 12 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 12 to 25 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 12 to 22 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 14 to 20 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 15 to 25 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 18 to 22 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 19 to 21 subunits in length. In certain embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 30, 18 to 50, 19 to 30, 19 to 50, or 20 to 30 linked subunits in length.

In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 12 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 13 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 14 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 15 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 16 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 17 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 18 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 19 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 20 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 21 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 22 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 23 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 24 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 25 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 26 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 27 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 28 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 29 subunits in length. In certain embodiments, an antisense compound targeted to a SOD-1 nucleic acid is 30 subunits in length. In certain embodiments, the antisense compound targeted to a SOD-1 nucleic acid is 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In certain embodiments the antisense compound is a modified oligonucleotide, and the linked subunits are nucleosides.

In certain embodiments, oligonucleotides targeted to a SOD-1 nucleic acid may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a SOD-1 nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.

It is possible to increase or decrease the length of an antisense compound, such as a modified oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase oligonucleotides, and a 28 and 42 nucleobase oligonucleotides comprised of the sequence of two or three of the tandem oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase oligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a SOD-1 nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE, and 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4′-(CH₂)_(n)—O-2′ bridge, where n=1 or n=2 and 4′-CH₂—O—CH₂-2′). In certain embodiments, wings may include several modified sugar moieties, including, for example 2′-MOE. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2′-MOE nucleosides and 2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as “X—Y—Z”, where “X” represents the length of the 5′ wing, “Y” represents the length of the gap, and “Z” represents the length of the 3′ wing. “X” and “Z” may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, “X” and “Y” may include one or more 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As used herein, a gapmer described as “X—Y—Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5′ wing and the 3′ wing. Thus, no intervening nucleotides exist between the 5′ wing and gap, or the gap and the 3′ wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, “X” and “Z” are the same; in other embodiments they are different.

In certain embodiments, gapmers provided herein include, for example 20-mers having a motif of 5-10-5.

In certain embodiments, gapmers provided herein include, for example 19-mers having a motif of 5-9-5.

In certain embodiments, gapmers provided herein include, for example 18-mers having a motif of 5-8-5.

In certain embodiments, gapmers provided herein include, for example 18-mers having a motif of 4-8-5.

In certain embodiments, gapmers provided herein include, for example 18-mers having a motif of 5-8-7.

In certain embodiments, gapmers provided herein include, for example 18-mers having a motif of 6-8-6.

In certain embodiments, gapmers provided herein include, for example 18-mers having a motif of 6-8-5.

In certain embodiments, the modified oligonucleotide contains at least one 2′-O-methoxyethyl modified nucleoside, at least one cEt modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the modified oligonucleotide has a sugar chemistry motif of any of the following:

-   -   ekddddddddekekee     -   kekeddddddddekek     -   eeeedddddddddkkee     -   eeeeddddddddekeke     -   eeeeddddddddkekee     -   eeeeddddddddkkeee     -   eeeeeddddddddkkee     -   eeeekddddddddkeee     -   eeeekdddddddkeeee     -   eeekddddddddkeeee     -   eeekkdddddddkkeee     -   eekkdddddddddkkee     -   eekkddddddddeeeee     -   eekkddddddddkkeee     -   ekekddddddddeeeee     -   ekekddddddddkekee     -   kekeddddddddeeeee, wherein     -   e=a 2′-O-methoxyethylribose modified sugar,     -   k=a cEt modified sugar,     -   d=a 2′-deoxyribose sugar,         Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode SOD-1 include, without limitation, the following: GENBANK Accession No. NM_000454.4 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000 to 18704000 (incorporated herein as SEQ ID NO: 2), and the complement of GENBANK Accession No. NW_001114168.1 truncated from nucleotides 2258000 to U.S. Pat. No. 2,271,000 (incorporated herein as SEQ ID NO: 3).

It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for SOD-1 can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, a target region may encompass the sequence from a 5′ target site of one target segment within the target region to a 3′ target site of another target segment within the same target region.

Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceeding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5′ target sites or 3′ target sites listed herein.

Suitable target segments may be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifically exclude a certain structurally defined region such as the start codon or stop codon.

The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In certain embodiments, reductions in SOD-1 mRNA levels are indicative of inhibition of SOD-1 expression. Reductions in levels of a SOD-1 protein are also indicative of inhibition of target mRNA expression. Phenotypic changes are indicative of inhibition of SOD-1 expression. Improvement in neurological function is indicative of inhibition of SOD-1 expression. Improved motor function is indicative of inhibition of SOD-1 expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a SOD-1 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a SOD-1 nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a SOD-1 nucleic acid).

Non-complementary nucleobases between an antisense compound and a SOD-1 nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a SOD-1 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a SOD-1 nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e., 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to a SOD-1 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e., linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a SOD-1 nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a SOD-1 nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

In certain embodiments, a portion of the oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

In certain embodiments, modified oligonucleotides targeted to a SOD-1 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are interspersed throughout the antisense compound. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of a modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, the modified oligonucleotides targeted to a SOD-1 nucleic acid comprise one or more phosphodiester internucleoside linkages. In certain embodiments, modified oligonucleotides targeted to a SOD-1 nucleic acid comprise at least one phosphorothioate internucleoside linkage and at least one phosphodiester internucleoside linkage. In certain embodiments, the modified oligonucleotide has a mixed backbone motif of the following:

-   -   sossssssssoooss,     -   sooossssssssoss,     -   sooosssssssssoss,     -   soosssssssssooss,     -   sooossssssssooss,     -   sooosssssssssooss,     -   sooossssssssssooss,     -   sooosssssssssssooos,     -   soooossssssssssooss,     -   sooosssssssssssooss,     -   sososssssssssssosos, and     -   sooossssssssssoooss, wherein     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.         Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R, R1 and R2 are each independently H, C₁-C₁₂ alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂OCH₃ substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R₁)—(CH₂)₂—N(R_(m))(R_(n)), where each R1, R_(m) and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleic acids (BNAs) include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof see PCT/US2008/068922 published as WO/2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof see PCT/US2008/064591 published as WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof see PCT/US2008/066154 published as WO 2008/154401, published on Dec. 8, 2008).

Further bicyclic nucleosides have been reported in published literature (see for example: Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wahlestedt et al., Proc. Nat. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; U.S. Pat. Nos. 7,399,845; 7,053,207; 7,034,133; 6,794,499; 6,770,748; 6,670,461; 6,525,191; 6,268,490; U.S. Patent Publication Nos.: US2008-0039618; US2007-0287831; US2004-0171570; U.S. patent application Ser. Nos. 12/129,154; 61/099,844; 61/097,787; 61/086,231; 61/056,564; 61/026,998; 61/026,995; 60/989,574; International applications WO 2007/134181; WO 2005/021570; WO 2004/106356; WO 94/14226; and PCT International Applications Nos.: PCT/US2008/068922; PCT/US2008/066154; and PCT/US2008/064591). Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

As used herein, “monocylic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ carbon atoms of the pentofuranosyl sugar moiety including without limitation, bridges comprising 1 or from 1 to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NRa)—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or a protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is, —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certain embodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′- wherein each R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-(CH₂)—O-2′ bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include those having a 4′ to 2′ bridge wherein such bridges include without limitation, α-L-4′-(CH₂)—O-2′, β-D-4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′, 4′-CH₂—N(R)—O-2′, 4′-CH(CH₃)—O-2′, 4′-CH₂—S-2′, 4′-CH₂—N(R)-2′, 4′-CH₂—CH(CH₃)-2′, and 4′-(CH₂)₃-2′, wherein R is H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiment, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—, —CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thiol.

In one embodiment, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃, OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) and J_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl and X is O or NJ_(c).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl or substituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl, substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl or substituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl.

The synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a 4′-CH₂—O-2′ bridge, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of bicyclic nucleosides has also been described in WO 98/39352 and WO 99/14226.

Analogs of various bicyclic nucleosides that have 4′ to 2′ bridging groups such as 4′-CH₂—O-2′ and 4′-CH₂—S-2′, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of oligodeoxyribonucleotide duplexes comprising bicyclic nucleosides for use as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel conformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and 2′-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl, substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k); and q_(i) and q_(j) or q_(i) and q_(k) together are ═C(q_(g))(q_(h)), wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and the alkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Frier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy (4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy (4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA, and (K) vinyl BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protecting group, C₁-C₆ alkyl or C₁-C₆ alkoxy.

As used herein, the term “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” substituted for the pentofuranosyl residue in normal nucleosides and can be referred to as a sugar surrogate. Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA) having a tetrahydropyranyl ring system as illustrated below.

In certain embodiment, sugar surrogates are selected having the formula:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the oligomeric compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an oligomeric compound or oligonucleotide and the other of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugate group or a 5′ or 3-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl; and

one of R₁ and R₂ is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides are provided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ is fluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following formula:

In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 published on Aug. 21, 2008 for other disclosed 5′, 2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvith et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenyl nucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugate group, or a 5′- or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugar substituent group.

Many other monocyclic, bicyclic and tricyclic ring systems are known in the art and are suitable as sugar surrogates that can be used to modify nucleosides for incorporation into oligomeric compounds as provided herein (see for example review article: Leumann, Christian J. Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to further enhance their activity.

As used herein, “2′-modified sugar” means a furanosyl sugar modified at the 2′ position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F, O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modified nucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2′-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

As used herein, “2′-modified” or “2′-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2′ position other than H or OH. 2′-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2′ substituents, such as allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. 2′-modified nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugar comprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃”, “2′-O-methyl” or “2′-methoxy” each refers to a nucleoside comprising a sugar comprising an —OCH₃ group at the 2′ position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or “2′-O-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative U.S. patents that teach the preparation of such modified sugars include without limitation, U.S. Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 on Dec. 22, 2005, and each of which is herein incorporated by reference in its entirety.

As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the (4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wings of a gapmer motif.

Compositions and Methods for Formulating Pharmaceutical Compositions

Oligonucleotides may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

An antisense compound targeted to a SOD-1 nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a SOD-1 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is a modified oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expression of SOD-1 nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.). Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, primary hepatocytes, A431 cells, and SH-SY5Y cells.

In Vitro Testing of Oligonucleotides

Described herein are methods for treatment of cells with oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.

Cells may be treated with oligonucleotides when the cells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of oligonucleotide and a LIPOFECTIN concentration that may range from 2 to 12 ug/mL per 100 nM oligonucleotide.

Another reagent used to introduce oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM oligonucleotide.

Another technique used to introduce oligonucleotides into cultured cells includes electroporation.

Cells are treated with oligonucleotides by routine methods. Cells may be harvested 16-24 hours after oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.

The concentration of oligonucleotide used varies from cell line to cell line. Methods to determine the optimal oligonucleotide concentration for a particular cell line are well known in the art. Oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of a SOD-1 nucleic acid can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents may be obtained from Invitrogen (Carlsbad, Calif.). RT real-time-PCR reactions are carried out by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.). Methods of RNA quantification by RIBOGREEN are SOD-1ght in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence.

Probes and primers are designed to hybridize to a SOD-1 nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of SOD-1 nucleic acids can be assessed by measuring SOD-1 protein levels. Protein levels of SOD-1 can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. In certain embodiments, the compounds herein provide improved reduction in protein levels.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, modified oligonucleotides, are tested in animals to assess their ability to inhibit expression of SOD-1 and produce phenotypic changes, such as, improved motor function. In certain embodiments, motor function is measured by walking initiation analysis, rotarod, grip strength, pole climb, open field performance, balance beam, hindpaw footprint testing in the animal. Testing may be performed in normal animals, or in experimental disease models. For administration to animals, oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Oligonucleotide dosage and dosing frequency depends upon multiple factors such as, but not limited to, route of administration and animal body weight. Following a period of treatment with oligonucleotides, RNA is isolated from CNS tissue or CSF and changes in SOD-1 nucleic acid expression are measured.

Certain Indications

In certain embodiments, provided herein are methods, compounds, and compositions of treating an individual comprising administering one or more pharmaceutical compositions described herein. In certain embodiments, the individual has a neurodegenerative disease. In certain embodiments, the individual is at risk for developing a neurodegenerative disease, including, but not limited to, amyotrophic lateral sclerosis (ALS). In certain embodiments, the individual has been identified as having a SOD-1 associated disease. In certain embodiments, provided herein are methods for prophylactically reducing SOD-1 expression in an individual. Certain embodiments include treating an individual in need thereof by administering to an individual a therapeutically effective amount of an antisense compound targeted to a SOD-1 nucleic acid.

In one embodiment, administration of a therapeutically effective amount of an antisense compound targeted to a SOD-1 nucleic acid is accompanied by monitoring of SOD-1 levels in an individual, to determine an individual's response to administration of the antisense compound. An individual's response to administration of the antisense compound may be used by a physician to determine the amount and duration of therapeutic intervention.

In certain embodiments, administration of an antisense compound targeted to a SOD-1 nucleic acid results in reduction of SOD-1 expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or a range defined by any two of these values. In certain embodiments, administration of an antisense compound targeted to a SOD-1 nucleic acid results in improved motor function in an animal. In certain embodiments, administration of a SOD-1 antisense compound improves motor function by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or a range defined by any two of these values.

In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to SOD-1 are used for the preparation of a medicament for treating a patient suffering or susceptible to a neurodegenerative disease including amyotrophic lateral sclerosis (ALS).

Certain Comparator Compositions

Antisense oligonucleotides targeting human SOD-1 were described in an earlier publication (see WO 2005/040180, incorporated by reference herein, in its entirety). Several oligonucleotides (ISIS 333611, ISIS 146144, ISIS 146145, ISIS 150437, ISIS 150441, ISIS 150443, ISIS 150444, ISIS 150445, ISIS 150446, ISIS 150447, ISIS 150448, ISIS 150449, ISIS 150452, ISIS 150454, ISIS 150458, ISIS 150460, ISIS 150462-150467, ISIS 150470, ISIS 150472, ISIS 150474, ISIS 150475, ISIS 150476, ISIS 150479-150483, ISIS 150488, ISIS 150489, ISIS 150490, ISIS 150491-150493, ISIS 150495-150498, ISIS 150511, ISIS 333605, ISIS 333606, ISIS 333609-333617, ISIS 333619, ISIS 333620-333636, ISIS 333638, and ISIS 333640) described therein, were used as comparator compounds throughout select screens for new antisense compounds described herein.

In certain embodiments, ISIS 333611, a 5-10-5 MOE gapmer, having a sequence of (from 5′ to 3′) CCGTCGCCCTTCAGCACGCA (incorporated herein as SEQ ID NO: 21), wherein each internucleoside linkage is a phosphorothioate linkage, each cytosine is a 5-methylcytosine, and each of nucleosides 1-5 and 16-20 (from 5′ to 3′) comprise a 2′-O-methoxyethyl moiety was used as a comparator compound. ISIS 333611 was selected as a comparator compound because it exhibited high levels of dose-dependent inhibition in various studies as described in WO 2005/040180. Additionally, phase 1 human clinical trials were completed using ISIS 333611. See, MILLER et al., “An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: a phase 1, randomised, first-in-man study” Lancet Neurol. (2013) 12(5): 435-442. Thus, ISIS 333611 was deemed a highly efficacious and potent compound with an acceptable safety profile (such that it was tested in human patients).

In certain embodiments, the compounds described herein benefit from one or more improved properties relative to the antisense compounds described in WO 2005/040180. Some of the improved properties are demonstrated in the examples provided herein. In certain embodiments, compounds described herein are more efficacious, potent, and/or tolerable in various in vitro and in vivo studies than comparator compounds described herein, including ISIS 333611. In certain embodiments, ISIS 666853, ISIS 666859, ISIS 666919, ISIS 666921, ISIS 666922, ISIS 666869, ISIS 666870, and ISIS 666867 are more efficacious and/or potent in various in vitro and in vivo studies than comparator compounds described herein, including ISIS 333611. In certain embodiments, ISIS 666853, ISIS 666859, ISIS 666919, ISIS 666921, ISIS 666922, ISIS 666869, ISIS 666870, and ISIS 666867 are more tolerable in one or more tolerability assays in animals than comparator compounds described herein, including ISIS 333611. This is despite 333611 being sufficiently well tolerated to progress to human clinical trials.

In certain embodiments, certain compounds described herein are more efficacious than comparator compounds by virtue of an in vitro IC50 of less than 2 μM, less than 1.9 μM, less than 1.8 μM, less than 1.7 μM, less than 1.6 μM, less than 1.5 μM, less than 1.4 μM, less than 1.3 μM, less than 1.2 μM, less than 1.1 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, or less than 0.5 μM less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, less than 0.1 μM, when tested in human cells, for example, in the HepG2 A431 or SH-SY5Y cell lines (For example, see Examples 6-11).

In certain embodiments, certain compounds described herein are more efficacious than comparator compounds by virtue of their ability to inhibit SOD-1 expression in vivo. In certain embodiments, the compounds inhibit SOD-1 in lumbar spinal cord and cervical spinal cord by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% in, for example, a transgenic animal model.

In certain embodiments, certain compounds described herein are more tolerable than comparator compounds on the basis of reduced microglial marker levels (e.g., IBA1), reduced astrocytic marker levels (e.g., GFAP), and/or FOB scores in rats, mice, and/or monkeys. See, for example, Examples 14, 15, 18, and 19.

ISIS 666853

For example, as provided in Example 12 (hereinbelow), ISIS 666853 achieved 81% inhibition in lumbar spinal cord and 74% in cervical spinal cord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67 mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose), whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and 47% inhibition in cervical spinal cord.

For example, as provided in Example 14 (hereinbelow), ISIS 666853 achieved a FOB score of 0 whereas ISIS 333611 achieved a FOB score of 4 in Sprague-Dawley rats after 3 hours when treated with 3 mg of oligonucleotide. Microglial marker (IBA1) levels and astrocytic marker (GFAP) levels were also reduced in ISIS 666853 treated rats as compared to ISIS 333611 treated rats.

For example, as provided in Example 15 (hereinbelow), ISIS 666853 achieved an ED₅₀ of 81.3 and 242.6 in lumbar tissue and cervical tissue (respectively) in SOD-1 transgenic rats when treated intrathecally with 10, 30, 100, 300, or 3000 μg of oligonucleotide. ED₅₀ in lumbar and cervical tissues could not be calculated in ISIS 333611 treated transgenic rats because the highest concentration tested (3000 μg) filed to inhibit human SOD-1 mRNA greater than 55-65%.

For example, as provided in Example 16 (hereinbelow), at doses of 1 mg and 3 mg ISIS 666853 achieved 3 hour FOB scores of 0.0 and 0.5 (respectively) whereas ISIS 333611 achieved FOB scores of 3.0 and 4.9 (respectively). At doses of 1 mg and 3 mg ISIS 666853 achieved 8 week FOB scores of 0.0 and 0.0 (respectively) whereas ISIS 333611 achieved FOB scores of 0.0 and 1.2 (respectively).

For example, as provided in Example 17 (hereinbelow), ISIS 666853 achieved an ED₅₀ of 136 and 188 in lumbar tissue and cortex tissue (respectively) whereas ISIS 333611 achieved an ED₅₀ of 401 and 786 in lumbar tissue and cortex tissue (respectively) in SOD-1 transgenic mice when treated with an intracerebral ventricular bolus of 10, 30, 100, 300, or 700 μg of oligonucleotide.

For example, as provided in Example 18 (hereinbelow), ISIS 666853 achieved a FOB score of 1.25 whereas ISIS 333611 achieved a FOB score of 6.5 in C57bl6 mice after 3 hours when treated with 700 μg of oligonucleotide. Microglial marker (IBA1) levels and astrocytic marker (GFAP) levels were also reduced in ISIS 666853 treated mice as compared to ISIS 333611 treated mice.

ISIS 666859

For example, as provided in Example 12 (hereinbelow), ISIS 666859 achieved 79% inhibition in lumbar spinal cord and 64% inhibition in cervical spinal cord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67 mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose), whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and 47% inhibition in cervical spinal cord.

For example, as provided in Example 14 (hereinbelow), ISIS 666859 achieved a FOB score of 1 whereas ISIS 333611 achieved a FOB score of 4 in Sprague-Dawley rats after 3 hours when treated with 3 mg of oligonucleotide. Microglial marker (IBA1) levels and astrocytic marker (GFAP) levels were also reduced in ISIS 666859 treated rats as compared to ISIS 333611 treated rats.

For example, as provided in Example 15 (hereinbelow), ISIS 666859 achieved an ED₅₀ of 74.0 and 358.8 in lumbar tissue and cervical tissue (respectively) in SOD-1 transgenic rats when treated intrathecally with 10, 30, 100, 300, or 3000 μg of oligonucleotide. ED₅₀ in lumbar and cervical tissues could not be calculated in ISIS 333611 treated transgenic rats because the highest concentration tested (3000 μg) filed to inhibit human SOD-1 mRNA greater than 55-65%.

For example, as provided in Example 16 (hereinbelow), at doses of 1 mg and 3 mg ISIS 666859 achieved 3 hour FOB scores of 0.0 and 2.1 (respectively) whereas ISIS 333611 achieved FOB scores of 3.0 and 4.9 (respectively). At doses of 1 mg and 3 mg ISIS 666859 achieved 8 week FOB scores of 0.0 and 0.3 (respectively) whereas ISIS 333611 achieved FOB scores of 0.0 and 1.2 (respectively).

For example, as provided in Example 17 (hereinbelow), ISIS 666859 achieved an ED₅₀ of 106 and 206 in lumbar tissue and cortex tissue (respectively) whereas ISIS 333611 achieved an ED₅₀ of 401 and 786 in lumbar tissue and cortex tissue (respectively) in SOD-1 transgenic mice when treated with an intracerebral ventricular bolus of 10, 30, 100, 300, or 700 μg of oligonucleotide.

For example, as provided in Example 18 (hereinbelow), ISIS 666859 achieved a FOB score of 1.75 whereas ISIS 333611 achieved a FOB score of 6.5 in C57bl6 mice after 3 hours when treated with 700 μg of oligonucleotide. Microglial marker (IBA1) levels and astrocytic marker (GFAP) levels were also reduced in ISIS 666859 treated mice as compared to ISIS 333611 treated mice.

ISIS 666919

For example, as provided in Example 12 (hereinbelow), ISIS 666919 achieved 76% inhibition in lumbar spinal cord and 68% in cervical spinal cord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67 mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose), whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and 47% inhibition in cervical spinal cord.

For example, as provided in Example 14 (hereinbelow), ISIS 666919 achieved a FOB score of 2 whereas ISIS 333611 achieved a FOB score of 4 in Sprague-Dawley rats after 3 hours when treated with 3 mg of oligonucleotide. Microglial marker (IBA1) levels and astrocytic marker (GFAP) levels were also reduced in ISIS 666919 treated rats as compared to ISIS 333611 treated rats.

For example, as provided in Example 15 (hereinbelow), ISIS 666919 achieved an ED₅₀ of 104.1 and 613.5 in lumbar tissue and cervical tissue (respectively) in SOD-1 transgenic rats when treated intrathecally with 10, 30, 100, 300, or 3000 μg of oligonucleotide. ED₅₀ in lumbar and cervical tissues could not be calculated in ISIS 333611 treated transgenic rats because the highest concentration tested (3000 μg) filed to inhibit human SOD-1 mRNA greater than 55-65%.

For example, as provided in Example 16 (hereinbelow), at doses of 1 mg and 3 mg ISIS 666919 achieved 3 hour FOB scores of 1.3 and 3.5 (respectively) whereas ISIS 333611 achieved FOB scores of 3.0 and 4.9 (respectively). At doses of 1 mg and 3 mg ISIS 666919 achieved 8 week FOB scores of 0.0 and 0.1 (respectively) whereas ISIS 333611 achieved FOB scores of 0.0 and 1.2 (respectively).

For example, as provided in Example 17 (hereinbelow), ISIS 666919 achieved an ED₅₀ of 168 in lumbar tissue whereas ISIS 333611 achieved an ED₅₀ of 401 in lumbar tissue in SOD-1 transgenic mice when treated with an intracerebral ventricular bolus of 10, 30, 100, 300, or 700 μg of oligonucleotide.

For example, as provided in Example 18 (hereinbelow), ISIS 666919 achieved a FOB score of 0.0 whereas ISIS 333611 achieved a FOB score of 6.5 in C57bl6 mice after 3 hours when treated with 700 μg of oligonucleotide. Microglial marker (IBA1) levels and astrocytic marker (GFAP) levels were also reduced in ISIS 666919 treated mice as compared to ISIS 333611 treated mice.

ISIS 666921

For example, as provided in Example 12 (hereinbelow), ISIS 66621 achieved 71% inhibition in lumbar spinal cord and 65% in cervical spinal cord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67 mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose), whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and 47% inhibition in cervical spinal cord.

For example, as provided in Example 14 (hereinbelow), ISIS 666921 achieved a FOB score of 2 whereas ISIS 333611 achieved a FOB score of 4 in Sprague-Dawley rats after 3 hours when treated with 3 mg of oligonucleotide. Microglial marker (IBA1) levels and astrocytic marker (GFAP) levels were also reduced in ISIS 666919 treated rats as compared to ISIS 333611 treated rats.

ISIS 666922

For example, as provided in Example 12 (hereinbelow), ISIS 666922 achieved 67% inhibition in lumbar spinal cord and 62% in cervical spinal cord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67 mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose), whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and 47% inhibition in cervical spinal cord.

For example, as provided in Example 14 (hereinbelow), ISIS 666922 achieved a FOB score of 3 whereas ISIS 333611 achieved a FOB score of 4 in Sprague-Dawley rats after 3 hours when treated with 3 mg of oligonucleotide. Microglial marker (IBA1) levels and astrocytic marker (GFAP) levels were also reduced in ISIS 666919 treated rats as compared to ISIS 333611 treated rats.

ISIS 666869

For example, as provided in Example 12 (hereinbelow), ISIS 666869 achieved 82% inhibition in lumbar spinal cord and 81% in cervical spinal cord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67 mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose), whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and 47% inhibition in cervical spinal cord.

ISIS 666870

For example, as provided in Example 12 (hereinbelow), ISIS 666870 achieved 76% inhibition in lumbar spinal cord and 68% in cervical spinal cord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67 mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose), whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and 47% inhibition in cervical spinal cord.

For example, as provided in Example 15 (hereinbelow), ISIS 666870 achieved an ED₅₀ of 139.4 and 1111 in lumbar tissue and cervical tissue (respectively) in SOD-1 transgenic rats when treated intrathecally with 10, 30, 100, 300, or 3000 μg of oligonucleotide. ED₅₀ in lumbar and cervical tissues could not be calculated in ISIS 333611 treated transgenic rats because the highest concentration tested (3000 μg) filed to inhibit human SOD-1 mRNA greater than 55-65%.

For example, as provided in Example 17 (hereinbelow), ISIS 666870 achieved an ED₅₀ of 148 and 409 in lumbar tissue and cortex tissue (respectively) whereas ISIS 333611 achieved an ED₅₀ of 401 and 786 in lumbar tissue and cortex tissue (respectively) in SOD-1 transgenic mice when treated with an intracerebral ventricular bolus of 10, 30, 100, 300, or 700 μg of oligonucleotide.

For example, as provided in Example 18 (hereinbelow), ISIS 666870 achieved a FOB score of 4.75 whereas ISIS 333611 achieved a FOB score of 6.5 in C57bl6 mice after 3 hours when treated with 700 μg of oligonucleotide.

ISIS 666867

For example, as provided in Example 12 (hereinbelow), ISIS 666867 achieved 59% inhibition in lumbar spinal cord and 48% in cervical spinal cord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67 mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose), whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and 47% inhibition in cervical spinal cord.

Certain Compositions

1. ISIS 666853

In certain embodiments, ISIS 666853 is characterized as a 5-10-5 MOE gapmer, having a sequence of (from 5′ to 3′) CAGGATACATTTCTACAGCT (incorporated herein as SEQ ID NO: 725), wherein each of nucleosides 1-5 and 16-20 are 2′-O-methoxyethylribose modified nucleosides, and each of nucleosides 6-15 are 2′-deoxynucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 4 to 5, 16 to 17, and 18 to 19 are phosphodiester linkages and the internucleoside linkages between nucleosides 1 to 2, 3 to 4, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 17 to 18, and 19 to 20 are phosphorothioate linkages, and wherein each cytosine is a 5′-methylcytosine.

In certain embodiments, ISIS 666853 is described by the following chemical notation: mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds Tds Tds mCds Tds Ads mCeo Aes Geo mCes Te; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666853 is described by the following chemical structure:

2. ISIS 666859

In certain embodiments, ISIS 666859 is characterized as a modified oligonucleotide having the nucleobase sequence (from 5′ to 3′) TTAATGTTTATCAGGAT (incorporated herein as SEQ ID NO: 1351), consisting of seventeen nucleosides, wherein each of nucleosides 1-4 and 15-17 are 2′-O-methoxyethylribose nucleosides, wherein each of nucleosides 13 and 14 are cEt modified nucleosides, wherein each of nucleosides 5-12 are 2′-deoxyribonucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 13 to 14, 14 to 15 are phosphodiester linkages and the internucleoside linkages between nucleosides 1 to 2, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 15 to 16, and 16 to 17 are phosphorothioate linkages, and wherein each cytosine is a 5′-methylcytosine.

In certain embodiments, ISIS 666859 is described by the following chemical notation: Tes Teo Aeo Aes Tds Gds Tds Tds Tds Ads Tds mCds Ako Gko Ges Aes Te; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666859 is described by the following chemical structure:

3. ISIS 666919

In certain embodiments, ISIS 666919 is characterized as a modified oligonucleotide having the nucleobase sequence (from 5′ to 3′) GGATACATTTCTACAGC (incorporated herein as SEQ ID NO: 1342), consisting of seventeen nucleosides, wherein each of nucleosides 1-4 and 16-17 are 2′-O-methoxyethylribose modified nucleosides, wherein each of nucleosides 14 and 15 are cEt modified nucleosides, wherein each of nucleosides 5-13 are 2′-deoxyribonucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, and 14 to 15 are phosphodiester linkages and the internucleoside linkages between nucleosides 1 to 2, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 15 to 16, and 16 to 17 are phosphorothioate linkages, and wherein each cytosine is a 5′-methylcytosine.

In certain embodiments, ISIS 666919 is described by the following chemical notation: Ges Geo Aeo Teo Ads mCds Ads Tds Tds Tds mCds Tds Ads mCko Aks Ges mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666919 is described by the following chemical structure:

4. ISIS 666921

In certain embodiments, ISIS 666921 is characterized as a modified oligonucleotide having the nucleobase sequence (from 5′ to 3′) GGATACATTTCTACAGC (incorporated herein as SEQ ID NO: 1342), consisting of seventeen nucleosides, wherein each of nucleosides 1-5 and 16-17 are 2′-O-methoxyethylribose modified nucleosides, wherein each of nucleosides 14-15 are cEt modified nucleosides, wherein each of nucleosides 6-13 are 2′-deoxyribonucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, and 14 to 15 are phosphodiester linkages and the internucleoside linkages between nucleosides 1 to 2, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 15 to 16, and 16 to 17 are phosphorothioate linkages, and wherein each cytosine is a 5′-methylcytosine.

In certain embodiments, ISIS 666921 is described by the following chemical notation: Ges Geo Aeo Teo Aes mCds Ads Tds Tds Tds mCds Tds Ads mCko Aks Ges mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666921 is described by the following chemical structure:

5. ISIS 666922

In certain embodiments, ISIS 666922 is characterized as a modified oligonucleotide having the nucleobase sequence (from 5′ to 3′) GGATACATTTCTACAGC (incorporated herein as SEQ ID NO: 1342), consisting of seventeen nucleosides, wherein each of nucleosides 1-4 and 15-17 are 2′-O-methoxyethylribose modified nucleosides, wherein each of nucleosides 5 and 14 are cEt modified nucleosides, wherein each of nucleosides 6-13 are 2′-deoxyribonucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, and 14 to 15 are phosphodiester linkages and the internucleoside linkages between nucleosides 1 to 2, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 15 to 16, and 16 to 17 are phosphorothioate linkages, and wherein each cytosine is a 5′-methylcytosine.

In certain embodiments, ISIS 666922 is described by the following chemical notation: Ges Geo Aeo Teo Aks mCds Ads Tds Tds Tds mCds Tds Ads mCko Aes Ges mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666922 is described by the following chemical structure:

6. ISIS 666869

In certain embodiments, ISIS 666869 is characterized as modified oligonucleotide having the nucleobase sequence (from 5′ to 3′) AGTGTTTAATGTTTATC (incorporated herein as SEQ ID NO: 1173), consisting of seventeen nucleosides, wherein each of nucleosides 1, 3, 14, and 16-17 are 2′-methoxyethylribose modified nucleosides, wherein each of nucleosides 2, 4, 13, and 15 are cEt modified nucleosides, wherein each of nucleosides 5-12 are 2′-deoxyribonucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 13 to 14, and 14 to 15 are phosphodiester linkages and the internucleoside linkages between nucleosides 1 to 2, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 15 to 16, and 16 to 17 are phosphorothioate linkages, and wherein each cytosine is a 5′-methylcytosine.

In certain embodiments, ISIS 666869 is described by the following chemical notation: Aes Gko Teo Gks Tds Tds Tds Ads Ads Tds Gds Tds Tko Teo Aks Tes mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666869 is described by the following chemical structure:

7. ISIS 666870

In certain embodiments, ISIS 666870 is characterized as a modified oligonucleotide having the nucleobase sequence (from 5′ to 3′) AGTGTTTAATGTTTATC (incorporated herein as SEQ ID NO: 1173), consisting of seventeen nucleosides, wherein each of nucleosides 1, 3, 13-17 are 2′-O-methoxyethylribose modified nucleosides, wherein each of nucleosides 2 and 4 are cEt modified nucleosides, wherein each of nucleosides 5-12 are 2′-deoxyribonucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 13 to 14, and 14 to 15 are phosphodiester linkages and the internucleoside linkages between nucleosides 1 to 2, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 15 to 16, and 16 to 17 are phosphorothioate linkages, and wherein each cytosine is a 5′-methylcytosine.

In certain embodiments, ISIS 666870 is described by the following chemical notation: Aes Gko Teo Gks Tds Tds Tds Ads Ads Tds Gds Tds Teo Teo Aes Tes mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666870 is described by the following chemical structure:

8. ISIS 666867

In certain embodiments, ISIS 666867 is characterized as a modified oligonucleotide having the nucleobase sequence (from 5′ to 3′) AGTGTTTAATGTTTATC (incorporated herein as SEQ ID NO: 1173), consisting of seventeen nucleosides, wherein each of nucleosides 1-2 and 13-17 are 2′-O-methoxyethylribose modified nucleosides, wherein each of nucleosides 3 and 4 are cEt modified nucleosides, wherein each of nucleosides 5-12 are 2′-deoxyribonucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 13 to 14, and 14 to 15 are phosphodiester linkages and the internucleoside linkages between nucleosides 1 to 2, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 15 to 16, and 16 to 17 are phosphorothioate linkages, and wherein each cytosine is a 5′-methylcytosine.

In certain embodiments, ISIS 666867 is described by the following chemical notation: Aes Geo Tko Gks Tds Tds Tds Ads Ads Tds Gds Tds Teo Teo Aes Tes mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666867 is described by the following chemical structure:

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions, and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.

Example 1: Inhibition of Human Superoxide Dismutase 1, Soluble (SOD-1) in HepG2 Cells by MOE Gapmers

Modified oligonucleotides were designed targeting a superoxide dismutase 1, soluble (SOD-1) nucleic acid and were tested for their effects on SOD-1 mRNA in vitro. ISIS 146144, ISIS 146145, ISIS 150437, ISIS 150441, ISIS 150443, ISIS 150444, ISIS 150445, ISIS 150446, ISIS 150447, ISIS 150448, ISIS 150449, ISIS 150452, ISIS 150454, ISIS 150458, ISIS 150460, ISIS 150462-150467, ISIS 150470, ISIS 150472, ISIS 150474, ISIS 150475, ISIS 150476, ISIS 150479-150483, ISIS 150488, ISIS 150489, ISIS 150490, ISIS 150491-150493, ISIS 150495-150498, ISIS 150511, ISIS 333605, ISIS 333606, ISIS 333609-333617, ISIS 333619, ISIS 333620-333636, ISIS 333638, and ISIS 333640, previously disclosed in WO 2005/040180, were also included in this assay. ISIS 333611, previously disclosed in WO 2005/040180, was also designated as a benchmark or comparator oligonucleotide. ISIS 333611 was recently tested in human clinical trials. See, MILLER et al., “An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: a phase 1, randomised, first-in-man study” Lancet Neurol. (2013) 12(5): 435-442.

The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 7,000 nM modified oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR.

Human primer probe set RTS3898 (forward sequence CTCTCAGGAGACCATTGCATCA, designated herein as SEQ ID NO: 11; reverse sequence TCCTGTCTTTGTACTTTCTTCATTTCC; designated herein as SEQ ID NO: 12; probe sequence CCGCACACTGGTGGTCCATGAAAA, designated herein as SEQ ID NO: 13) was used to measure mRNA levels. In cases where the oligonucleotide overlapped the amplicon of the primer probe set, an alternative primer probe set, HTS90 (forward sequence CGTGGCCTAGCGAGTTATGG, designated herein as SEQ ID NO: 14; reverse sequence GAAATTGATGATGCCCTGCA; designated herein as SEQ ID NO: 15; probe sequence ACGAAGGCCGTGTGCGTGCTGX, designated herein as SEQ ID NO: 16), was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells. ‘n.d.’ indicates that inhibition levels were not measured using the particular primer probe set.

The newly designed modified oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment is comprised of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ directions comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Startsite” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stopsite” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human SOD-1 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the human SOD-1 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000 to 18704000). ‘n/a’ indicates that the modified oligonucleotide does not target that particular gene sequence with 100 complementarity.

TABLE 1 Percent Inhibition of SOD-1 mRNA by 5-10-5 MOE  gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: % NO: NO: 1 1 inhibition 2 2 SEQ ISIS Start Stop with Start Stop ID NO Site Site Sequence RTS3898 Site Site NO 590065 1 20 CGCCCACTCTGGCCCCAAAC 7 807 826 118 590066 35 54 CCGCGACTACTTTATAGGCC 5 841 860 119 333611 167 186 CCGTCGCCCTTCAGCACGCA 85 973 992 21 590067 202 221 CCTTCTGCTCGAAATTGATG 74 1008 1027 120 590068 203 222 TCCTTCTGCTCGAAATTGAT 58 n/a n/a 121 590069 204 223 TTCCTTCTGCTCGAAATTGA 50 n/a n/a 122 590070 205 224 TTTCCTTCTGCTCGAAATTG 47 n/a n/a 123 590071 206 225 CTTTCCTTCTGCTCGAAATT 31 n/a n/a 124 590072 207 226 ACTTTCCTTCTGCTCGAAAT 42 n/a n/a 125 590073 208 227 TACTTTCCTTCTGCTCGAAA 38 n/a n/a 126 590074 209 228 TTACTTTCCTTCTGCTCGAA 33 n/a n/a 127 590075 210 229 ATTACTTTCCTTCTGCTCGA 39 n/a n/a 128 590076 211 230 CATTACTTTCCTTCTGCTCG 28 n/a n/a 129 590077 212 231 CCATTACTTTCCTTCTGCTC 58 n/a n/a 130 590078 213 232 TCCATTACTTTCCTTCTGCT 58 n/a n/a 131 590079 214 233 GTCCATTACTTTCCTTCTGC 69 n/a n/a 132 590080 215 234 GGTCCATTACTTTCCTTCTG 68 n/a n/a 133 590081 216 235 TGGTCCATTACTTTCCTTCT 61 n/a n/a 134 590082 217 236 CTGGTCCATTACTTTCCTTC 69 n/a n/a 135 590083 218 237 ACTGGTCCATTACTTTCCTT 54 4972 4991 136 150445 219 238 CACTGGTCCATTACTTTCCT 84 4973 4992 22 590084 220 239 TCACTGGTCCATTACTTTCC 65 4974 4993 137 590085 221 240 TTCACTGGTCCATTACTTTC 45 4975 4994 138 590086 222 241 CTTCACTGGTCCATTACTTT 43 4976 4995 139 590087 223 242 CCTTCACTGGTCCATTACTT 67 4977 4996 140 590088 224 243 ACCTTCACTGGTCCATTACT 59 4978 4997 141 436841 225 244 CACCTTCACTGGTCCATTAC 65 4979 4998 142 150446 226 245 ACACCTTCACTGGTCCATTA 83 4980 4999 23 393336 227 246 CACACCTTCACTGGTCCATT 81 4981 5000 143 150447 228 247 CCACACCTTCACTGGTCCAT 89 4982 5001 24 590089 229 248 CCCACACCTTCACTGGTCCA 82 4983 5002 144 590090 230 249 CCCCACACCTTCACTGGTCC 89 4984 5003 145 590091 231 250 TCCCCACACCTTCACTGGTC 84 4985 5004 146 590092 232 251 TTCCCCACACCTTCACTGGT 61 4986 5005 147 590093 233 252 CTTCCCCACACCTTCACTGG 60 4987 5006 148 590094 234 253 GCTTCCCCACACCTTCACTG 78 4988 5007 149 590095 235 254 TGCTTCCCCACACCTTCACT 72 4989 5008 150 590096 236 255 ATGCTTCCCCACACCTTCAC 76 4990 5009 151 393337 237 256 AATGCTTCCCCACACCTTCA 76 4991 5010 152 590097 238 257 TAATGCTTCCCCACACCTTC 68 4992 5011 153 590098 264 283 TCCATGCAGGCCTTCAGTCA 63 5018 5037 154 590099 265 284 ATCCATGCAGGCCTTCAGTC 64 5019 5038 155 590100 266 285 AATCCATGCAGGCCTTCAGT 52 5020 5039 156 590101 267 286 GAATCCATGCAGGCCTTCAG 53 5021 5040 157 590102 268 287 GGAATCCATGCAGGCCTTCA 65 5022 5041 158 393339 269 288 TGGAATCCATGCAGGCCTTC 43 5023 5042 159 590103 270 289 ATGGAATCCATGCAGGCCTT 56 5024 5043 160 590104 271 290 CATGGAATCCATGCAGGCCT 57 5025 5044 161 590105 272 291 ACATGGAATCCATGCAGGCC 52 5026 5045 162 590106 273 292 AACATGGAATCCATGCAGGC 54 5027 5046 163 590107 274 293 GAACATGGAATCCATGCAGG 51 5028 5047 164 590108 275 294 TGAACATGGAATCCATGCAG 58 5029 5048 165 393340 276 295 ATGAACATGGAATCCATGCA 62 5030 5049 166 590109 316 335 GACCTGCACTGGTACAGCCT 69 7632 7651 167 436847 317 336 GGACCTGCACTGGTACAGCC 74 7633 7652 168 590110 318 337 AGGACCTGCACTGGTACAGC 70 7634 7653 169 590111 319 338 GAGGACCTGCACTGGTACAG 74 7635 7654 170 590112 320 339 TGAGGACCTGCACTGGTACA 68 7636 7655 171 590113 321 340 GTGAGGACCTGCACTGGTAC 80 7637 7656 172 393343 322 341 AGTGAGGACCTGCACTGGTA 79 7638 7657 173 590114 323 342 AAGTGAGGACCTGCACTGGT 65 7639 7658 174 590115 324 343 AAAGTGAGGACCTGCACTGG 48 7640 7659 175 590116 325 344 TAAAGTGAGGACCTGCACTG 51 7641 7660 176 436848 326 345 TTAAAGTGAGGACCTGCACT 59 7642 7661 177 590117 327 346 ATTAAAGTGAGGACCTGCAC 43 7643 7662 178 590118 328 347 GATTAAAGTGAGGACCTGCA 43 7644 7663 179 590119 329 348 GGATTAAAGTGAGGACCTGC 67 7645 7664 180 590120 330 349 AGGATTAAAGTGAGGACCTG 63 7646 7665 181 436849 331 350 GAGGATTAAAGTGAGGACCT 64 7647 7666 182 393344 332 351 AGAGGATTAAAGTGAGGACC 59 7648 7667 183 590121 333 352 TAGAGGATTAAAGTGAGGAC 52 7649 7668 184 590122 334 353 ATAGAGGATTAAAGTGAGGA 36 7650 7669 185 590123 335 354 GATAGAGGATTAAAGTGAGG 25 7651 7670 186 590124 336 355 GGATAGAGGATTAAAGTGAG 34 7652 7671 187 590125 337 356 TGGATAGAGGATTAAAGTGA 49 7653 7672 188 590126 338 357 CTGGATAGAGGATTAAAGTG 34 7654 7673 189 590127 339 358 TCTGGATAGAGGATTAAAGT 39 7655 7674 190 590128 360 379 ATCCTTTGGCCCACCGTGTT 60 7676 7695 191

TABLE 2 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: % % NO: NO: 1 1 inhibition inhibition 2 2 SEQ ISIS Start Stop with with Start Stop ID NO Site Site Sequence RTS3898 HTS90 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA 76 95  973  992 21 393347 361 380 CATCCTTTGGCCCACCGTGT 70 72 7677 7696 192 590129 362 381 TCATCCTTTGGCCCACCGTG 66 68 7678 7697 193 590130 363 382 TTCATCCTTTGGCCCACCGT 53 55 7679 7698 194 590131 364 383 CTTCATCCTTTGGCCCACCG 52 50 7680 7699 195 590132 365 384 TCTTCATCCTTTGGCCCACC 61 64 7681 7700 196 590133 366 385 CTCTTCATCCTTTGGCCCAC 45 54 7682 7701 197 590134 367 386 TCTCTTCATCCTTTGGCCCA 44 34 7683 7702 198 590135 368 387 CTCTCTTCATCCTTTGGCCC 52 49 7684 7703 199 590136 369 388 CCTCTCTTCATCCTTTGGCC 48 47 7685 7704 200 590137 370 389 GCCTCTCTTCATCCTTTGGC 35 44 n/a n/a 201 590138 371 390 TGCCTCTCTTCATCCTTTGG 52 45 n/a n/a 202 590139 372 391 ATGCCTCTCTTCATCCTTTG 50 45 n/a n/a 203 590140 373 392 CATGCCTCTCTTCATCCTTT 49 27 n/a n/a 204 590141 374 393 ACATGCCTCTCTTCATCCTT 34 18 n/a n/a 205 590142 375 394 AACATGCCTCTCTTCATCCT 38 35 n/a n/a 206 333612 376 395 CAACATGCCTCTCTTCATCC 34 33 n/a n/a 25 333613 377 396 CCAACATGCCTCTCTTCATC 46 55 n/a n/a 26 333614 378 397 TCCAACATGCCTCTCTTCAT 42 48 n/a n/a 27 333615 379 398 CTCCAACATGCCTCTCTTCA 42 15 n/a n/a 28 333616 380 399 TCTCCAACATGCCTCTCTTC 35 44 n/a n/a 29 333617 381 400 GTCTCCAACATGCCTCTCTT 42 47 n/a n/a 30 590143 501 520 TGCTTTTTCATGGACCACCA n.d. 65 n/a n/a 207 590144 502 521 CTGCTTTTTCATGGACCACC n.d. 70 n/a n/a 208 590145 503 522 TCTGCTTTTTCATGGACCAC n.d. 64 n/a n/a 209 436860 504 523 ATCTGCTTTTTCATGGACCA n.d. 65 n/a n/a 210 590146 505 524 CATCTGCTTTTTCATGGACC n.d. 68 9655 9674 211 590147 506 525 TCATCTGCTTTTTCATGGAC n.d. 59 9656 9675 212 393359 507 526 GTCATCTGCTTTTTCATGGA n.d. 56 9657 9676 213 590148 508 527 AGTCATCTGCTTTTTCATGG n.d. 45 9658 9677 214 590149 509 528 AAGTCATCTGCTTTTTCATG n.d. 23 9659 9678 215 590150 510 529 CAAGTCATCTGCTTTTTCAT n.d. 43 9660 9679 216 590151 511 530 CCAAGTCATCTGCTTTTTCA n.d. 72 9661 9680 217 489513 512 531 CCCAAGTCATCTGCTTTTTC n.d. 73 9662 9681 218 590152 513 532 GCCCAAGTCATCTGCTTTTT n.d. 74 9663 9682 219 436861 514 533 TGCCCAAGTCATCTGCTTTT n.d. 75 9664 9683 220 590153 515 534 TTGCCCAAGTCATCTGCTTT n.d. 47 9665 9684 221 393360 516 535 TTTGCCCAAGTCATCTGCTT n.d. 57 9666 9685 222 590154 517 536 CTTTGCCCAAGTCATCTGCT n.d. 79 9667 9686 223 590155 518 537 CCTTTGCCCAAGTCATCTGC n.d. 67 9668 9687 224 590156 519 538 ACCTTTGCCCAAGTCATCTG n.d. 57 9669 9688 225 333620 520 539 CACCTTTGCCCAAGTCATCT n.d. 68 9670 9689 31 333621 521 540 CCACCTTTGCCCAAGTCATC n.d. 72 9671 9690 32 333622 522 541 TCCACCTTTGCCCAAGTCAT n.d. 77 9672 9691 33 333623 523 542 TTCCACCTTTGCCCAAGTCA n.d. 73 9673 9692 34 333624 524 543 TTTCCACCTTTGCCCAAGTC n.d. 77 9674 9693 35 333625 525 544 ATTTCCACCTTTGCCCAAGT n.d. 79 9675 9694 36 333626 526 545 CATTTCCACCTTTGCCCAAG n.d. 72 9676 9695 37 333627 527 546 TCATTTCCACCTTTGCCCAA n.d. 55 9677 9696 38 333628 528 547 TTCATTTCCACCTTTGCCCA n.d. 59 9678 9697 39 333629 529 548 CTTCATTTCCACCTTTGCCC n.d. 73 9679 9698 40 333630 530 549 TCTTCATTTCCACCTTTGCC n.d. 76 9680 9699 41 333631 531 550 TTCTTCATTTCCACCTTTGC n.d. 62 9681 9700 42 333632 532 551 TTTCTTCATTTCCACCTTTG n.d. 64 9682 9701 43 333633 533 552 CTTTCTTCATTTCCACCTTT n.d. 69 9683 9702 44 333634 534 553 ACTTTCTTCATTTCCACCTT n.d. 55 9684 9703 45 333635 535 554 TACTTTCTTCATTTCCACCT n.d. 72 9685 9704 46 489517 582 601 CCCAATTACACCACAAGCCA 68 72 9732 9751 226 436863 583 602 TCCCAATTACACCACAAGCC 83 86 9733 9752 227 590157 584 603 ATCCCAATTACACCACAAGC 64 62 9734 9753 228 590158 585 604 GATCCCAATTACACCACAAG 51 61 9735 9754 229 590159 586 605 CGATCCCAATTACACCACAA 60 55 9736 9755 230 590160 587 606 GCGATCCCAATTACACCACA 59 63 9737 9756 231 150463 588 607 GGCGATCCCAATTACACCAC 78 79 9738 9757 47 393363 589 608 GGGCGATCCCAATTACACCA 65 65 9739 9758 232 590161 590 609 TGGGCGATCCCAATTACACC 56 60 9740 9759 233 590162 591 610 TTGGGCGATCCCAATTACAC 48 51 9741 9760 234 489518 592 611 ATTGGGCGATCCCAATTACA 51 59 9742 9761 235 436864 593 612 TATTGGGCGATCCCAATTAC 39 41 9743 9762 236 590163 594 613 TTATTGGGCGATCCCAATTA 35 34 9744 9763 237 590164 595 614 TTTATTGGGCGATCCCAATT 42 44 9745 9764 238 590165 596 615 GTTTATTGGGCGATCCCAAT 58 61 9746 9765 239 393364 597 616 TGTTTATTGGGCGATCCCAA 60 69 9747 9766 240 590166 598 617 ATGTTTATTGGGCGATCCCA 51 54 9748 9767 241 590167 599 618 AATGTTTATTGGGCGATCCC 48 45 9749 9768 242 590168 600 619 GAATGTTTATTGGGCGATCC 60 65 9750 9769 243 150464 601 620 GGAATGTTTATTGGGCGATC 58 63 9751 9770 48 393365 607 626 TCCAAGGGAATGTTTATTGG 50 58 9757 9776 244

TABLE 3 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE  gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: % NO: NO: 1 1 inhibition 2 2 SEQ ISIS Start Stop with Start Stop ID NO Site Site Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA 80 973 992 21 590169 608 627 ATCCAAGGGAATGTTTATTG 63 9758 9777 245 590170 609 628 CATCCAAGGGAATGTTTATT 53 9759 9778 246 590171 610 629 ACATCCAAGGGAATGTTTAT 49 9760 9779 247 590172 611 630 TACATCCAAGGGAATGTTTA 56 9761 9780 248 489519 612 631 CTACATCCAAGGGAATGTTT 60 9762 9781 249 590173 613 632 ACTACATCCAAGGGAATGTT 61 9763 9782 250 590174 614 633 GACTACATCCAAGGGAATGT 65 9764 9783 251 393366 615 634 AGACTACATCCAAGGGAATG 58 9765 9784 252 590175 616 635 CAGACTACATCCAAGGGAAT 50 9766 9785 253 436865 617 636 TCAGACTACATCCAAGGGAA 69 9767 9786 254 590176 618 637 CTCAGACTACATCCAAGGGA 78 9768 9787 255 150465 619 638 CCTCAGACTACATCCAAGGG 91 9769 9788 49 590177 620 639 GCCTCAGACTACATCCAAGG 90 9770 9789 256 590178 621 640 GGCCTCAGACTACATCCAAG 92 9771 9790 257 489520 622 641 GGGCCTCAGACTACATCCAA 88 9772 9791 258 590179 643 662 CAGGATAACAGATGAGTTAA 79 9793 9812 259 590180 644 663 GCAGGATAACAGATGAGTTA 83 9794 9813 260 590181 645 664 AGCAGGATAACAGATGAGTT 81 9795 9814 261 590182 646 665 TAGCAGGATAACAGATGAGT 68 9796 9815 262 590183 647 666 CTAGCAGGATAACAGATGAG 74 9797 9816 263 590184 648 667 GCTAGCAGGATAACAGATGA 70 9798 9817 264 393370 649 668 AGCTAGCAGGATAACAGATG 61 9799 9818 265 590185 650 669 CAGCTAGCAGGATAACAGAT 78 9800 9819 266 590186 651 670 ACAGCTAGCAGGATAACAGA 72 9801 9820 267 489522 652 671 TACAGCTAGCAGGATAACAG 78 9802 9821 268 590187 653 672 CTACAGCTAGCAGGATAACA 88 9803 9822 269 378879 654 673 TCTACAGCTAGCAGGATAAC 86 9804 9823 270 590188 655 674 TTCTACAGCTAGCAGGATAA 85 9805 9824 271 393371 656 675 TTTCTACAGCTAGCAGGATA 84 9806 9825 272 436868 657 676 ATTTCTACAGCTAGCAGGAT 81 9807 9826 273 590189 658 677 CATTTCTACAGCTAGCAGGA 87 9808 9827 274 590190 659 678 ACATTTCTACAGCTAGCAGG 92 9809 9828 275 590191 660 679 TACATTTCTACAGCTAGCAG 88 9810 9829 276 590192 661 680 ATACATTTCTACAGCTAGCA 88 9811 9830 277 489523 662 681 GATACATTTCTACAGCTAGC 93 9812 9831 278 590193 683 702 ACAGTGTTTAATGTTTATCA 74 9833 9852 279 590194 684 703 TACAGTGTTTAATGTTTATC 64 9834 9853 280 590195 685 704 TTACAGTGTTTAATGTTTAT 56 9835 9854 281 590196 686 705 ATTACAGTGTTTAATGTTTA 50 9836 9855 282 590197 687 706 GATTACAGTGTTTAATGTTT 74 9837 9856 283 590198 688 707 AGATTACAGTGTTTAATGTT 37 9838 9857 284 590199 689 708 AAGATTACAGTGTTTAATGT 58 9839 9858 285 393375 690 709 TAAGATTACAGTGTTTAATG 58 9840 9859 286 590200 691 710 TTAAGATTACAGTGTTTAAT 46 9841 9860 287 436876 772 791 CAAATCTTCCAAGTGATCAT 36 9922 9941 288 590201 773 792 ACAAATCTTCCAAGTGATCA 33 9923 9942 289 590202 774 793 TACAAATCTTCCAAGTGATC 34 9924 9943 290 150474 775 794 ATACAAATCTTCCAAGTGAT 47 9925 9944 50 590203 776 795 TATACAAATCTTCCAAGTGA 29 9926 9945 291 393382 777 796 CTATACAAATCTTCCAAGTG 41 9927 9946 292 436877 778 797 ACTATACAAATCTTCCAAGT 45 9928 9947 293 590204 779 798 AACTATACAAATCTTCCAAG 27 9929 9948 294 590205 780 799 AAACTATACAAATCTTCCAA 33 9930 9949 295 590206 781 800 AAAACTATACAAATCTTCCA 35 9931 9950 296 489533 782 801 TAAAACTATACAAATCTTCC 26 9932 9951 297 590207 783 802 ATAAAACTATACAAATCTTC 19 9933 9952 298 590208 784 803 TATAAAACTATACAAATCTT 2 9934 9953 299 590209 785 804 TTATAAAACTATACAAATCT 7 9935 9954 300 590210 786 805 TTTATAAAACTATACAAATC 0 9936 9955 301 590211 787 806 TTTTATAAAACTATACAAAT 4 9937 9956 302 590212 788 807 GTTTTATAAAACTATACAAA 5 9938 9957 303 590213 789 808 AGTTTTATAAAACTATACAA 3 9939 9958 304 436878 790 809 GAGTTTTATAAAACTATACA 7 9940 9959 305 150475 791 810 TGAGTTTTATAAAACTATAC 28 9941 9960 51 489536 812 831 CATTGAAACAGACATTTTAA 28 9962 9981 306 150479 813 832 TCATTGAAACAGACATTTTA 36 9963 9982 52 393385 814 833 GTCATTGAAACAGACATTTT 50 9964 9983 307 590214 815 834 GGTCATTGAAACAGACATTT 45 9965 9984 308 590215 816 835 AGGTCATTGAAACAGACATT 47 9966 9985 309 590216 817 836 CAGGTCATTGAAACAGACAT 39 9967 9986 310 590217 818 837 ACAGGTCATTGAAACAGACA 44 9968 9987 311 590218 819 838 TACAGGTCATTGAAACAGAC 42 9969 9988 312 150480 820 839 ATACAGGTCATTGAAACAGA 46 9970 9989 53 393386 821 840 AATACAGGTCATTGAAACAG 36 9971 9990 313 489537 822 841 AAATACAGGTCATTGAAACA 12 9972 9991 314 590219 823 842 AAAATACAGGTCATTGAAAC 16 9973 9992 315 590220 824 843 CAAAATACAGGTCATTGAAA 21 9974 9993 316

TABLE 4 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ ID ID SEQ SEQ NO: NO: % ID ID 1 1 inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO Site Site Sequence RTS3898 Site Site NO 590251 n/a n/a CCTTGCCTTCTGCTCGAAAT 57 1013 1032 317 590252 n/a n/a AATAAAGTTGACCTCTTTTT 45 5479 5498 318 590253 n/a n/a CTCTGATATAAAAATCTTGT 54 8142 8161 319 590254 n/a n/a GCCCCGCGGCGGCCTCGGTC 38 1238 1257 320 590255 n/a n/a GCTATCGCCATTATTACAAG 38 7722 7741 321 590256 n/a n/a CTCAAATGTGAAAGTTGTCC 57 3414 3433 322 590257 n/a n/a GTTCTATATTCAATAAATGC 37 7925 7944 323 590258 n/a n/a AATTAAAGTTCCCAAATACA 0 7578 7597 324 590259 n/a n/a GATCATTACAAAAGTTAAGA 17 6150 6169 325 590260 n/a n/a CCTTCTCTGCCCTTGCAGCC 55 1685 1704 326 590261 n/a n/a ACCCAAATAACTATGTTGTA n.d. 9394 9413 327 590262 n/a n/a CCAGGTTTTAAACTTAACAA n.d. 8890 8909 328 590263 n/a n/a ATCTCAGGACTAAAATAAAC 44 3663 3682 329 590264 n/a n/a AAATAACTATGTTGTAGACC n.d. 9390 9409 330 590265 n/a n/a AAGAACCTTTTCCAGAAAAT 37 2449 2468 331 590266 n/a n/a GGAACAGAAACAAGTCTATG 25 7458 7477 332 590267 n/a n/a AGAAAGCTATCGCCATTATT 27 7727 7746 333 590268 n/a n/a TTCCCAAATACATTCTAAAA 7 7570 7589 334 590269 n/a n/a AACTGCTCTAGGCCTGTGTC 53 4787 4806 335 590270 n/a n/a AAATGGATCAAATCTGATCA 31 6595 6614 336 590271 n/a n/a GTAGGTGCACATCAAAATCA 58 1928 1947 337 590272 n/a n/a TCTGATATAAAAATCTTGTC 28 8141 8160 338 590273 n/a n/a ACCATATGAACTCCAGAAAG 45 7741 7760 339 590274 n/a n/a AACATCAAGGTAGTTCATGA 10 8379 8398 340 590275 n/a n/a GCAATTACAGAAATGGATCA 42 6605 6624 341 590276 n/a n/a TTTTAAGCATATTCCAAAGT 45 6331 6350 342 590277 n/a n/a TCAACCCCCAGCTCAAACAC 26 6174 6193 343 590278 n/a n/a AGAAAAATAACATTAATCCT n.d. 9541 9560 344 590279 n/a n/a AAGATTTTAAACACGGAATA 31 3085 3104 345 146145 165 184 GTCGCCCTTCAGCACGCACA 82 971 990 54 333611 167 186 CCGTCGCCCTTCAGCACGCA 81 973 992 21 590250 399 418 AGCAGTCACATTGCCCAAGT 75 8454 8473 346 489525 682 701 CAGTGTTTAATGTTTATCAG 69 9832 9851 347 436879 825 844 GCAAAATACAGGTCATTGAA 49 9975 9994 348 590221 826 845 GGCAAAATACAGGTCATTGA 54 9976 9995 349 590222 827 846 TGGCAAAATACAGGTCATTG 52 9977 9996 350 393387 828 847 CTGGCAAAATACAGGTCATT 51 9978 9997 351 590223 829 848 TCTGGCAAAATACAGGTCAT 47 9979 9998 352 590224 830 849 GTCTGGCAAAATACAGGTCA 44 9980 9999 353 590225 831 850 AGTCTGGCAAAATACAGGTC 50 9981 10000 354 489538 832 851 AAGTCTGGCAAAATACAGGT 38 9982 10001 355 590226 833 852 TAAGTCTGGCAAAATACAGG 33 9983 10002 356 590227 834 853 TTAAGTCTGGCAAAATACAG 20 9984 10003 357 150482 853 872 TTTAATACCCATCTGTGATT 29 10003 10022 55 590228 854 873 GTTTAATACCCATCTGTGAT 33 10004 10023 358 150483 855 874 AGTTTAATACCCATCTGTGA 44 10005 10024 56 590229 856 875 AAGTTTAATACCCATCTGTG 51 10006 10025 359 590230 857 876 CAAGTTTAATACCCATCTGT 42 10007 10026 360 590231 858 877 ACAAGTTTAATACCCATCTG 38 10008 10027 361 393389 859 878 GACAAGTTTAATACCCATCT 48 10009 10028 362 590232 860 879 TGACAAGTTTAATACCCATC 55 10010 10029 363 590233 861 880 CTGACAAGTTTAATACCCAT 49 10011 10030 364 489541 862 881 TCTGACAAGTTTAATACCCA 52 10012 10031 365 590234 863 882 TTCTGACAAGTTTAATACCC 39 10013 10032 366 590235 864 883 ATTCTGACAAGTTTAATACC 21 10014 10033 367 590236 865 884 AATTCTGACAAGTTTAATAC 4 10015 10034 368 393390 866 885 AAATTCTGACAAGTTTAATA 7 10016 10035 369 590237 867 886 GAAATTCTGACAAGTTTAAT 5 10017 10036 370 436881 868 887 AGAAATTCTGACAAGTTTAA 33 10018 10037 371 590238 869 888 AAGAAATTCTGACAAGTTTA 20 10019 10038 372 590239 891 910 TTATTCACAGGCTTGAATGA 23 10041 10060 373 489544 892 911 TTTATTCACAGGCTTGAATG 41 10042 10061 374 590240 893 912 TTTTATTCACAGGCTTGAAT 40 10043 10062 375 436884 894 913 TTTTTATTCACAGGCTTGAA 31 10044 10063 376 590241 895 914 GTTTTTATTCACAGGCTTGA 39 10045 10064 377 150488 896 915 GGTTTTTATTCACAGGCTTG 51 10046 10065 57 590242 897 916 GGGTTTTTATTCACAGGCTT 46 10047 10066 378 150489 898 917 AGGGTTTTTATTCACAGGCT 52 10048 10067 58 590243 899 918 CAGGGTTTTTATTCACAGGC 49 10049 10068 379 590244 900 919 ACAGGGTTTTTATTCACAGG 38 10050 10069 380 590245 901 920 TACAGGGTTTTTATTCACAG 34 10051 10070 381 150490 902 921 ATACAGGGTTTTTATTCACA 30 10052 10071 59 590246 903 922 CATACAGGGTTTTTATTCAC 34 10053 10072 382 150491 904 923 CCATACAGGGTTTTTATTCA 34 10054 10073 60 590247 905 924 GCCATACAGGGTTTTTATTC 34 10055 10074 383 590248 906 925 TGCCATACAGGGTTTTTATT 33 10056 10075 384 393393 907 926 GTGCCATACAGGGTTTTTAT 43 10057 10076 385 590249 908 927 AGTGCCATACAGGGTTTTTA 12 10058 10077 386

TABLE 5 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: % NO: NO: 1 1 inhibition 2 2 SEQ ISIS Start Stop with Start Stop ID NO Site Site Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA 86 973 992 21 590280 n/a n/a TGGAAAAACTCAAATGTGAA 51 3422 3441 387 590281 n/a n/a TTTCCCTTTCTTTTCCACAC 76 5738 5757 388 590282 n/a n/a TCTTTCCCTTTCTTTTCCAC 65 5740 5759 389 590283 n/a n/a TACCTTCTCTGCCCTTGCAG 74 1687 1706 390 590284 n/a n/a GCAAGGGCCAAGGCTGCTGC 75 6879 6898 391 590285 n/a n/a AAAGCTAAATTATGAATTAA 12 7592 7611 392 590286 n/a n/a CTAATGAAGGCTCAGTATGA 59 3193 3212 393 590287 n/a n/a GGAGTCAAATGCCAAAGAAC 60 2463 2482 394 590288 n/a n/a TGAATTAAAGTTCCCAAATA 5 7580 7599 395 590289 n/a n/a ACTTGGTGCAGGCAGAATAT 63 6916 6935 396 590290 n/a n/a CCTCTGATATAAAAATCTTG 67 8143 8162 397 590291 n/a n/a AAAGTTGGAGAGAGTTTCTG 8 4940 4959 398 590292 n/a n/a TCTCTGCCCTTGCAGCCCAA 80 1682 1701 399 590293 n/a n/a TTACTTGGTGCAGGCAGAAT 56 6918 6937 400 590294 n/a n/a AATGGAGTCAAATGCCAAAG 66 2466 2485 401 590295 n/a n/a TATGAATTAAAGTTCCCAAA 20 7582 7601 402 590296 n/a n/a AGTTCTATATTCAATAAATG 21 7926 7945 403 590297 n/a n/a TACAAGTAGTATACCATATG 33 7753 7772 404 590298 n/a n/a TAGCCTTAGAGCTGTACAAA 70 1553 1572 405 590299 n/a n/a GTCCCCATTTGTCAATTCCT 71 7882 7901 406 590300 n/a n/a AACCTGCCTACTGGCAGAGC 59 2095 2114 407 590301 n/a n/a CTTGTTCCCACACTCAATGC 56 4747 4766 408 590302 n/a n/a ACAAGTCATGATAACCTGCA 61 8952 8971 409 590303 n/a n/a TGTTTTCCAAACTCAGATCT 52 8796 8815 410 590304 n/a n/a AGAACCTCATAATATTAGAA 9 9557 9576 411 590305 n/a n/a GGTTTTAAACTTAACAAAAT 1 8887 8906 412 590306 n/a n/a CTCTGGTGTATTTTTAGTAA 65 1831 1850 413 590307 n/a n/a TATCTCTGCATATCTGGAAA 71 3034 3053 414 590308 n/a n/a CAGCCTTTTTAACCCAAAAG 68 4407 4426 415 590309 n/a n/a TGGAATGCTCCACTATCCAA 57 3012 3031 416 590310 n/a n/a CGTTCAGAAGTTTGTCTCTG 67 2126 2145 417 590311 n/a n/a CTGCTCAGGGAAGGTGGAAA 53 2922 2941 418 590312 n/a n/a TCAAGAGAAGCTAGGAAAAC 50 3154 3173 419 590313 n/a n/a TCCCTTTCTTTTCCACACCT 74 5736 5755 420 590314 n/a n/a TTGTTCCCACACTCAATGCA 56 4746 4765 421 590315 n/a n/a TCACCAGCACAGCACAACAC 58 5076 5095 422 590316 n/a n/a CCTGGGATCATTACAAAAGT 42 6155 6174 423 590317 n/a n/a AGTAGTATACCATATGAACT 35 7749 7768 424 590318 n/a n/a TCTAATATGGTCAAATGTAA 27 8779 8798 425 590319 n/a n/a GGTTGGGCTCTGGTGTATTT 64 1838 1857 426 590320 n/a n/a TGCCCTTTACTTGGTGCAGG 56 6924 6943 427 590321 n/a n/a AGAGAGTTTCTGAACAAAGA 24 4932 4951 428 590322 n/a n/a GAATTTCAGCAATTACAGAA 33 6613 6632 429 590323 n/a n/a ACAAGTTAAACAAGTCATGA 9 8961 8980 430 590324 n/a n/a TGTGCCCTTTACTTGGTGCA 47 6926 6945 431 590325 n/a n/a TTAGGAGGAGGAAAAGGACC 23 1719 1738 432 590326 n/a n/a ACTGGCAGAGCAATTTTAAA 25 2086 2105 433 590327 n/a n/a AGTCAAATGCCAAAGAACCT 58 2461 2480 434 590328 n/a n/a AAGCATCAGATGGATTAGGG 17 8411 8430 435 590329 n/a n/a GTCCGCGGGACCCTCAGGAA 54 1414 1433 436 590330 n/a n/a CAATTACAGAAATGGATCAA 42 6604 6623 437 590331 n/a n/a GCTGTCAAGTAATCACTACC 27 9606 9625 438 590332 n/a n/a AGTGCAAAGTTGGAGAGAGT 33 4945 4964 439 590333 n/a n/a ACTTGCTTCCAATCCCAAAT 78 6436 6455 440 590334 n/a n/a AACTCAAATGTGAAAGTTGT 51 3416 3435 441 590335 n/a n/a TTTTAGTAAGATCTTCAAAT 14 1820 1839 442 590336 n/a n/a ATTTCAGCAATTACAGAAAT 27 6611 6630 443 590337 n/a n/a TTAAGTGTCCCCATTTGTCA 56 7888 7907 444 590338 n/a n/a TTAGCAACCTGCCTACTGGC 57 2100 2119 445 590339 n/a n/a TATTACAAGAGTTAAGCATC 41 7711 7730 446 590340 n/a n/a ATGTTGAATATACATGTACA 36 4545 4564 447 590341 n/a n/a TTTGTCTCTGACCATCTTAG 74 2116 2135 448 590342 n/a n/a TTTTCCACCAGTTGGTAACT 59 2253 2272 449 590343 n/a n/a CAACAGCTTCCCACAAGTTA 28 8973 8992 450 590344 n/a n/a CAAATGTGAAAGTTGTCCCT 62 3412 3431 451 590345 n/a n/a GCTACCTTCTCTGCCCTTGC 73 1689 1708 452 590346 n/a n/a TCTTAGCAGAACAGTGTTCT 51 8743 8762 453 590347 n/a n/a ATACATTCTAAAAAGAAACA 41 7563 7582 454 590348 n/a n/a GCACATATTTACAAGTAGTA 58 7762 7781 455 590349 n/a n/a GGGTCACCAGCACAGCACAA 35 5079 5098 456 590350 n/a n/a GTGCAAGGGCCAAGGCTGCT 66 6881 6900 457 590351 n/a n/a ACCTGGGTTCATGCATGGAT 72 2902 2921 458 590352 n/a n/a ATCACTATTTGAAACTAAAT 0 6569 6588 459 590353 n/a n/a ATACAATAAAGTTGACCTCT 64 5483 5502 460 590354 n/a n/a TTTTAAACTTAACAAAATGT 10 8885 8904 461 590355 n/a n/a CTCCCCGCGCTCCCGCCACG 15 1268 1287 462 590356 n/a n/a GAAGGCTCAGTATGAAGAGA 65 3188 3207 463

TABLE 6  Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: % NO: NO: 1 1 inhibition 2 2 SEQ ISIS Start Stop with Start Stop ID NO Site Site Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA 87 973 992 21 590357 n/a n/a AGAAAACAGCTGATTTACCT 40 4915 4934 464 590358 n/a n/a CCACAAGTTAAACAAGTCAT n.d. 8963 8982 465 590359 n/a n/a CAAATTTGCAAACAAGTAGC 61 8331 8350 466 590360 n/a n/a CCTAATTTGAACTGCAAGTA n.d. 8665 8684 467 590361 n/a n/a AAAAAACTCATCTCCCCAGC 70 6969 6988 468 590362 n/a n/a AGGCTCAGTATGAAGAGATC 67 3186 3205 469 590363 n/a n/a TGTTATCAAGAGCACAGGGC 58 3383 3402 470 590364 n/a n/a CCTCAAAAGGGAGATGGTAA 41 4768 4787 471 590365 n/a n/a AGTATGGGTCACCAGCACAG 71 5084 5103 472 590366 n/a n/a TCACAATCTAGTGCAGTTAC 70 5584 5603 473 590367 n/a n/a CAAGTGAGAAACCCAATCCT n.d. 8856 8875 474 590368 n/a n/a AGAAAATCTGGCCATTTTAA n.d. 8832 8851 475 590369 n/a n/a ACAGGTAATGGTGCTCCGTG 71 3716 3735 476 590370 n/a n/a TGAAAGGCTTTCAGAAAACA 44 8102 8121 477 590371 n/a n/a CAGGCAAGTTACAGGAAGCA 64 6687 6706 478 590372 n/a n/a CAGCAAGCTGCTTAACTGCT 65 4800 4819 479 590373 n/a n/a TGTTGCAAAGACATTACCTT n.d. 9455 9474 480 590374 n/a n/a GAAACTAAATTAGCAAGATG 43 6559 6578 481 590375 n/a n/a TCAAGAGCACAGGGCCAAAA 60 3378 3397 482 590376 n/a n/a AGGAGGAGGAAAAGGACCTC 53 1717 1736 483 590377 n/a n/a CCTCAGCCTTTTTAACCCAA 73 4410 4429 484 590378 n/a n/a CTATGTTGTAGACCACCACA n.d. 9384 9403 485 590379 n/a n/a CTCCGTGGCTACATACAGAA 66 3703 3722 486 590380 n/a n/a TTTATCTGGATCTTTAGAAA n.d. 8642 8661 487 590381 n/a n/a AAAAAAAGGAAAGTGAAAGT n.d. 9279 9298 488 590382 n/a n/a GGTTCATGCATGGATTCTCA 76 2897 2916 489 590383 n/a n/a CTGCAAAGTGTCACACAAAC 76 1630 1649 490 590384 n/a n/a TTCAGAAGTACCAAAGGGTA 53 8227 8246 491 590385 n/a n/a TAAAAGCATTCCAGCATTTG 44 7848 7867 492 590386 n/a n/a TAGTATACCATATGAACTCC 73 7747 7766 493 590387 n/a n/a TGCATATCTGGAAAGCTGGA 59 3028 3047 494 590388 n/a n/a CTTAACTGCTCTAGGCCTGT 54 4790 4809 495 590389 n/a n/a AGGCACCGACCGGGCGGCAC 21 1155 1174 496 590390 n/a n/a TGCAAAGTTGGAGAGAGTTT 32 4943 4962 497 590391 n/a n/a TCCTCAAAAGGGAGATGGTA 37 4769 4788 498 590392 n/a n/a AGTATACCATATGAACTCCA 76 7746 7765 499 590393 n/a n/a TATTTGTACATGTTGAATAT 2 4554 4573 500 590394 n/a n/a ACCCAAAAGGTGTATGTCTC 71 4396 4415 501 590395 n/a n/a CTTTGGAAAAAAAGGAAAGT n.d. 9285 9304 502 590396 n/a n/a GGGAGAAAGGCAGGCAAGTT 20 6697 6716 503 590397 n/a n/a TTAAGCCCAGGAAGTAAAAG 9 7862 7881 504 590398 n/a n/a AGACATTACCTTTAAACATT n.d. 9447 9466 505 590399 n/a n/a GTGGCTTAAGAAATGCTCCG 26 2050 2069 506 590400 n/a n/a GTGAGAAGGGAACAGAAACA 48 7466 7485 507 590401 n/a n/a AAAAGCATCAGATGGATTAG 21 8413 8432 508 590402 n/a n/a TTCCACCAGTTGGTAACTTC 78 2251 2270 509 590403 n/a n/a TTTTTAGTAAGATCTTCAAA 15 1821 1840 510 590404 n/a n/a ATCTGTGTCCAAATCCCAGG 59 4847 4866 511 590405 n/a n/a TAAGATCTTCAAATAAGCTA 33 1814 1833 512 590406 n/a n/a ATCAACTCTTTCCCTTTCTT 63 5746 5765 513 590407 n/a n/a TGTGTCCTCAAAAGGGAGAT 37 4773 4792 514 590408 n/a n/a TACCTCCTCCCAACAATACC n.d. 9590 9609 515 590409 n/a n/a TTCTGCTTTACAACTATGGC n.d. 9133 9152 516 590410 n/a n/a GTACATGTTGAATATACATG 35 4549 4568 517 590411 n/a n/a TTTGTGGCTAATCTTAAGGT 47 5699 5718 518 590412 n/a n/a TCCTGCCTCAGCCTTTTTAA 34 4415 4434 519 590413 n/a n/a CGGTGTCCGCGGGACCCTCA 59 1418 1437 520 590414 n/a n/a GAAATGGATCAAATCTGATC 50 6596 6615 521 590415 n/a n/a GGTAGTTCATGAGCTAAATT 31 8371 8390 522 590416 n/a n/a AATGGAGTCTCGACTAGTTT 62 8072 8091 523 590417 n/a n/a CAAGTATGGGTCACCAGCAC 57 5086 5105 524 590418 n/a n/a GGTGTCCGCGGGACCCTCAG 40 1417 1436 525 590419 n/a n/a CGCCACGCGCAGGCCCAGCC 37 1255 1274 526 590420 n/a n/a TCTAGGCCTGTGTCCTCAAA 75 4781 4800 527 590421 n/a n/a ACTGTCCTGGGCTAATGAAG 36 3204 3223 528 590422 n/a n/a AAGCATCTTGTTACCTCTCT 52 7698 7717 529 590423 n/a n/a GCCCAGGAAGTAAAAGCATT 38 7858 7877 530 590424 n/a n/a GTAAGATCTTCAAATAAGCT 46 1815 1834 531 590425 n/a n/a AAAGGGAGATGGTAATCTTG 48 4763 4782 532 590426 n/a n/a GCCAAGGCTGCTGCCTTACA 66 6873 6892 533 590427 n/a n/a CAGACTAACTGTTCCTGTCC 43 2363 2382 534 590428 n/a n/a TTTGTCAATTCCTTTAAGCC 39 7875 7894 535 590429 n/a n/a ACTACCTCCTCCCAACAATA n.d. 9592 9611 536 590430 n/a n/a TACCTCTCTTCATCCTTTGG 50 7687 7706 537 590431 n/a n/a ACTGCTCTAGGCCTGTGTCC 59 4786 4805 538 590432 n/a n/a CCTCCTCCCAACAATACCCA n.d. 9588 9607 539 590433 n/a n/a GGCAGGCAAGTTACAGGAAG 42 6689 6708 540

TABLE 7  Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: % NO: NO: 1 1 inhibition 2 2 SEQ ISIS Start Stop with Start Stop ID NO Site Site Sequence RTS3898 Site Site NO 592596 2 21 TCGCCCACTCTGGCCCCAAA 86 808 827 541 592597 4 23 CCTCGCCCACTCTGGCCCCA 89 810 829 542 592598 6 25 CGCCTCGCCCACTCTGGCCC 56 812 831 543 592599 8 27 CGCGCCTCGCCCACTCTGGC 68 814 833 544 592600 10 29 TCCGCGCCTCGCCCACTCTG 64 816 835 545 592601 12 31 CCTCCGCGCCTCGCCCACTC 83 818 837 546 592602 14 33 GACCTCCGCGCCTCGCCCAC 89 820 839 547 592603 16 35 CAGACCTCCGCGCCTCGCCC 88 822 841 548 592604 18 37 GCCAGACCTCCGCGCCTCGC 79 824 843 549 592605 20 39 AGGCCAGACCTCCGCGCCTC 89 826 845 550 592606 22 41 ATAGGCCAGACCTCCGCGCC 88 828 847 551 592607 24 43 TTATAGGCCAGACCTCCGCG 75 830 849 552 592608 26 45 CTTTATAGGCCAGACCTCCG 21 832 851 553 592609 28 47 TACTTTATAGGCCAGACCTC 76 834 853 554 592610 30 49 ACTACTTTATAGGCCAGACC 60 836 855 555 592611 32 51 CGACTACTTTATAGGCCAGA 0 838 857 556 592612 34 53 CGCGACTACTTTATAGGCCA 0 840 859 557 592613 36 55 TCCGCGACTACTTTATAGGC 0 842 861 558 592614 38 57 TCTCCGCGACTACTTTATAG 7 844 863 559 592615 40 59 CGTCTCCGCGACTACTTTAT 0 846 865 560 592616 42 61 CCCGTCTCCGCGACTACTTT 0 848 867 561 592617 44 63 ACCCCGTCTCCGCGACTACT 0 850 869 562 592618 46 65 GCACCCCGTCTCCGCGACTA 0 852 871 563 592619 48 67 CAGCACCCCGTCTCCGCGAC 0 854 873 564 592620 50 69 ACCAGCACCCCGTCTCCGCG 0 856 875 565 592621 52 71 AAACCAGCACCCCGTCTCCG 2 858 877 566 592622 54 73 GCAAACCAGCACCCCGTCTC 4 860 879 567 592623 56 75 ACGCAAACCAGCACCCCGTC 0 862 881 568 592624 58 77 CGACGCAAACCAGCACCCCG 0 864 883 569 592625 60 79 TACGACGCAAACCAGCACCC 0 866 885 570 592626 62 81 ACTACGACGCAAACCAGCAC 0 868 887 571 592627 64 83 AGACTACGACGCAAACCAGC 1 870 889 572 592628 66 85 GGAGACTACGACGCAAACCA 0 872 891 573 592629 68 87 CAGGAGACTACGACGCAAAC 0 874 893 574 592630 70 89 TGCAGGAGACTACGACGCAA 0 876 895 575 592631 72 91 GCTGCAGGAGACTACGACGC 1 878 897 576 150511 74 93 ACGCTGCAGGAGACTACGAC 0 880 899 61 592632 90 109 GCAACGGAAACCCCAGACGC 2 896 915 577 592633 92 111 CTGCAACGGAAACCCCAGAC 0 898 917 578 592634 94 113 GACTGCAACGGAAACCCCAG 0 900 919 579 345715 95 114 GGACTGCAACGGAAACCCCA 0 901 920 580 592635 96 115 AGGACTGCAACGGAAACCCC 1 902 921 581 150437 98 117 CGAGGACTGCAACGGAAACC 6 904 923 62 592636 100 119 TCCGAGGACTGCAACGGAAA 6 906 925 582 592637 102 121 GTTCCGAGGACTGCAACGGA 12 908 927 583 592638 104 123 TGGTTCCGAGGACTGCAACG 0 910 929 584 592639 106 125 CCTGGTTCCGAGGACTGCAA 32 912 931 585 592640 108 127 GTCCTGGTTCCGAGGACTGC 68 914 933 586 345717 110 129 AGGTCCTGGTTCCGAGGACT 65 916 935 587 592641 112 131 CGAGGTCCTGGTTCCGAGGA 84 918 937 588 592642 114 133 GCCGAGGTCCTGGTTCCGAG 86 920 939 589 592643 116 135 ACGCCGAGGTCCTGGTTCCG 78 922 941 590 592644 118 137 CCACGCCGAGGTCCTGGTTC 79 924 943 591 345719 120 139 GGCCACGCCGAGGTCCTGGT 63 926 945 592 150441 122 141 TAGGCCACGCCGAGGTCCTG 81 928 947 63 592645 124 143 GCTAGGCCACGCCGAGGTCC 63 930 949 593 592646 126 145 TCGCTAGGCCACGCCGAGGT 56 932 951 594 592647 128 147 ACTCGCTAGGCCACGCCGAG 48 934 953 595 345721 130 149 TAACTCGCTAGGCCACGCCG 63 936 955 596 592648 132 151 CATAACTCGCTAGGCCACGC 38 938 957 597 592649 134 153 GCCATAACTCGCTAGGCCAC 52 940 959 598 592650 136 155 TCGCCATAACTCGCTAGGCC 59 942 961 599 592651 138 157 CGTCGCCATAACTCGCTAGG 55 944 963 600 592652 156 175 CAGCACGCACACGGCCTTCG 56 962 981 601 333605 158 177 TTCAGCACGCACACGGCCTT 85 964 983 64 333606 160 179 CCTTCAGCACGCACACGGCC 82 966 985 65 146144 162 181 GCCCTTCAGCACGCACACGG 58 968 987 66 333609 164 183 TCGCCCTTCAGCACGCACAC 79 970 989 67 146145 165 184 GTCGCCCTTCAGCACGCACA 86 971 990 54 333610 166 185 CGTCGCCCTTCAGCACGCAC 79 972 991 68 333611 167 186 CCGTCGCCCTTCAGCACGCA 83 973 992 21 592653 168 187 GCCGTCGCCCTTCAGCACGC 79 974 993 602 592654 169 188 GGCCGTCGCCCTTCAGCACG 72 975 994 603 592655 170 189 GGGCCGTCGCCCTTCAGCAC 51 976 995 604 592656 172 191 CTGGGCCGTCGCCCTTCAGC 45 978 997 605 592657 174 193 CACTGGGCCGTCGCCCTTCA 33 980 999 606 592658 176 195 TGCACTGGGCCGTCGCCCTT 72 982 1001 607 592659 178 197 CCTGCACTGGGCCGTCGCCC 76 984 1003 608

TABLE 8  Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: % NO: NO: 1 1 inhibition 2 2 SEQ ISIS Start Stop with Start Stop ID NO Site Site Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA 87 973 992 21 150443 180 199 GCCCTGCACTGGGCCGTCGC 65 986 1005 69 592660 182 201 ATGCCCTGCACTGGGCCGTC 52 988 1007 609 592661 184 203 TGATGCCCTGCACTGGGCCG 30 990 1009 610 592662 186 205 GATGATGCCCTGCACTGGGC 38 992 1011 611 592663 188 207 TTGATGATGCCCTGCACTGG 36 994 1013 612 150444 190 209 AATTGATGATGCCCTGCACT 48 996 1015 70 592664 192 211 GAAATTGATGATGCCCTGCA 35 998 1017 15 592665 194 213 TCGAAATTGATGATGCCCTG 40 1000 1019 614 592666 196 215 GCTCGAAATTGATGATGCCC 68 1002 1021 615 592667 198 217 CTGCTCGAAATTGATGATGC 63 1004 1023 616 592668 200 219 TTCTGCTCGAAATTGATGAT 47 1006 1025 617 592669 239 258 TTAATGCTTCCCCACACCTT 68 4993 5012 618 592670 241 260 CTTTAATGCTTCCCCACACC 71 4995 5014 619 592671 243 262 TCCTTTAATGCTTCCCCACA 69 4997 5016 620 150448 245 264 AGTCCTTTAATGCTTCCCCA 76 4999 5018 71 592672 247 266 TCAGTCCTTTAATGCTTCCC 75 5001 5020 621 592673 249 268 AGTCAGTCCTTTAATGCTTC 58 5003 5022 622 592674 251 270 TCAGTCAGTCCTTTAATGCT 46 5005 5024 623 592675 253 272 CTTCAGTCAGTCCTTTAATG 41 5007 5026 624 592676 255 274 GCCTTCAGTCAGTCCTTTAA 62 5009 5028 625 150449 257 276 AGGCCTTCAGTCAGTCCTTT 65 5011 5030 72 592677 259 278 GCAGGCCTTCAGTCAGTCCT 69 5013 5032 626 592678 261 280 ATGCAGGCCTTCAGTCAGTC 65 5015 5034 627 592679 263 282 CCATGCAGGCCTTCAGTCAG 53 5017 5036 628 592680 277 296 CATGAACATGGAATCCATGC 63 5031 5050 629 592681 279 298 CTCATGAACATGGAATCCAT 60 5033 5052 630 592682 281 300 AACTCATGAACATGGAATCC 56 5035 5054 631 592683 284 303 CCAAACTCATGAACATGGAA 60 5038 5057 632 592684 286 305 CTCCAAACTCATGAACATGG 69 5040 5059 633 592685 288 307 ATCTCCAAACTCATGAACAT 40 5042 5061 634 592686 290 309 TTATCTCCAAACTCATGAAC 35 5044 5063 635 592687 292 311 TATTATCTCCAAACTCATGA 26 5046 5065 636 592688 294 313 TGTATTATCTCCAAACTCAT 41 5048 5067 637 150452 296 315 GCTGTATTATCTCCAAACTC 51 5050 5069 73 592689 298 317 CTGCTGTATTATCTCCAAAC 43 5052 5071 638 592690 300 319 GCCTGCTGTATTATCTCCAA 37 n/a n/a 639 592691 302 321 CAGCCTGCTGTATTATCTCC 33 n/a n/a 640 592692 304 323 TACAGCCTGCTGTATTATCT 27 n/a n/a 641 592693 306 325 GGTACAGCCTGCTGTATTAT 21 n/a n/a 642 592694 308 327 CTGGTACAGCCTGCTGTATT 23 n/a n/a 643 592695 310 329 CACTGGTACAGCCTGCTGTA 46 n/a n/a 644 592696 312 331 TGCACTGGTACAGCCTGCTG 40 n/a n/a 645 592697 314 333 CCTGCACTGGTACAGCCTGC 62 n/a n/a 646 592698 340 359 TTCTGGATAGAGGATTAAAG 41 7656 7675 647 592699 342 361 TTTTCTGGATAGAGGATTAA 29 7658 7677 648 592700 344 363 TGTTTTCTGGATAGAGGATT 51 7660 7679 649 592701 346 365 CGTGTTTTCTGGATAGAGGA 64 7662 7681 650 592702 348 367 ACCGTGTTTTCTGGATAGAG 44 7664 7683 651 592703 350 369 CCACCGTGTTTTCTGGATAG 62 7666 7685 652 592704 352 371 GCCCACCGTGTTTTCTGGAT 60 7668 7687 653 592705 354 373 TGGCCCACCGTGTTTTCTGG 62 7670 7689 654 592706 356 375 TTTGGCCCACCGTGTTTTCT 49 7672 7691 655 592707 358 377 CCTTTGGCCCACCGTGTTTT 52 7674 7693 656 592708 382 401 AGTCTCCAACATGCCTCTCT 53 n/a n/a 657 592709 384 403 CAAGTCTCCAACATGCCTCT 39 n/a n/a 658 489501 386 405 CCCAAGTCTCCAACATGCCT 75 8441 8460 659 150454 388 407 TGCCCAAGTCTCCAACATGC 86 8443 8462 74 592710 390 409 ATTGCCCAAGTCTCCAACAT 71 8445 8464 660 592711 392 411 ACATTGCCCAAGTCTCCAAC 64 8447 8466 661 592712 394 413 TCACATTGCCCAAGTCTCCA 59 8449 8468 662 489502 396 415 AGTCACATTGCCCAAGTCTC 70 8451 8470 663 592713 398 417 GCAGTCACATTGCCCAAGTC 70 8453 8472 664 592714 400 419 CAGCAGTCACATTGCCCAAG 84 8455 8474 665 592715 402 421 GTCAGCAGTCACATTGCCCA 83 8457 8476 666 592716 404 423 TTGTCAGCAGTCACATTGCC 59 8459 8478 667 489503 406 425 CTTTGTCAGCAGTCACATTG 47 8461 8480 668 592717 408 427 ATCTTTGTCAGCAGTCACAT 54 8463 8482 669 592718 410 429 CCATCTTTGTCAGCAGTCAC 76 8465 8484 670 592719 412 431 CACCATCTTTGTCAGCAGTC 75 8467 8486 671 592720 414 433 CACACCATCTTTGTCAGCAG 66 8469 8488 672 489504 416 435 GCCACACCATCTTTGTCAGC 60 8471 8490 673 592721 418 437 CGGCCACACCATCTTTGTCA 62 8473 8492 674 592722 420 439 ATCGGCCACACCATCTTTGT 57 8475 8494 675 592723 422 441 ACATCGGCCACACCATCTTT 54 8477 8496 676 150458 424 443 ACACATCGGCCACACCATCT 77 8479 8498 75 489505 426 445 AGACACATCGGCCACACCAT 84 8481 8500 677 592724 428 447 ATAGACACATCGGCCACACC 66 8483 8502 678

TABLE 9  Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: % % NO: NO: 1 1 inhibition inhibition 2 2 SEQ ISIS Start Stop with with Start Stop ID NO Site Site Sequence RTS3898 HTS90 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA 87 n.d. 973 992 21 592725 430 449 CAATAGACACATCGGCCACA 67 65 8485 8504 679 592726 432 451 TTCAATAGACACATCGGCCA 62 66 8487 8506 680 592727 434 453 TCTTCAATAGACACATCGGC 56 49 8489 8508 681 489506 436 455 AATCTTCAATAGACACATCG 25 28 8491 8510 682 592728 438 457 AGAATCTTCAATAGACACAT 12 0 8493 8512 683 592729 440 459 ACAGAATCTTCAATAGACAC 24 16 8495 8514 684 592730 442 461 TCACAGAATCTTCAATAGAC 34 24 8497 8516 685 592731 444 463 GATCACAGAATCTTCAATAG 15 14 8499 8518 686 489507 446 465 GAGATCACAGAATCTTCAAT 42 46 8501 8520 687 592732 448 467 GTGAGATCACAGAATCTTCA n.d. 58 8503 8522 688 592733 450 469 GAGTGAGATCACAGAATCTT n.d. 45 8505 8524 689 592734 452 471 GAGAGTGAGATCACAGAATC n.d. 48 8507 8526 690 592735 454 473 CTGAGAGTGAGATCACAGAA n.d. 66 8509 8528 691 489508 456 475 TCCTGAGAGTGAGATCACAG n.d. 60 8511 8530 692 333619 458 477 TCTCCTGAGAGTGAGATCAC n.d. 65 8513 8532 76 592736 460 479 GGTCTCCTGAGAGTGAGATC n.d. 40 8515 8534 693 592737 462 481 ATGGTCTCCTGAGAGTGAGA n.d. 37 8517 8536 694 592738 464 483 CAATGGTCTCCTGAGAGTGA n.d. 41 8519 8538 695 489509 466 485 TGCAATGGTCTCCTGAGAGT n.d. 43 8521 8540 696 592739 468 487 GATGCAATGGTCTCCTGAGA n.d. 16 8523 8542 697 592740 470 489 ATGATGCAATGGTCTCCTGA n.d. 6 8525 8544 698 592741 472 491 CAATGATGCAATGGTCTCCT n.d. 0 8527 8546 699 592742 474 493 GCCAATGATGCAATGGTCTC n.d. 25 8529 8548 700 489510 476 495 CGGCCAATGATGCAATGGTC n.d. 32 8531 8550 701 592743 478 497 TGCGGCCAATGATGCAATGG n.d. 14 8533 8552 702 592744 480 499 TGTGCGGCCAATGATGCAAT n.d. 0 8535 8554 703 592745 482 501 AGTGTGCGGCCAATGATGCA n.d. 7 8537 8556 704 592746 484 503 CCAGTGTGCGGCCAATGATG n.d. 25 8539 8558 705 489511 486 505 CACCAGTGTGCGGCCAATGA n.d. 28 8541 8560 706 150460 488 507 ACCACCAGTGTGCGGCCAAT n.d. 52 8543 8562 77 592747 490 509 GGACCACCAGTGTGCGGCCA n.d. 44 n/a n/a 707 592748 492 511 ATGGACCACCAGTGTGCGGC n.d. 40 n/a n/a 708 150462 494 513 TCATGGACCACCAGTGTGCG n.d. 39 n/a n/a 78 592749 496 515 TTTCATGGACCACCAGTGTG n.d. 35 n/a n/a 709 592750 498 517 TTTTTCATGGACCACCAGTG n.d. 23 n/a n/a 710 592751 500 519 GCTTTTTCATGGACCACCAG n.d. 63 n/a n/a 711 333636 536 555 GTACTTTCTTCATTTCCACC n.d. 65 9686 9705 79 333638 538 557 TTGTACTTTCTTCATTTCCA n.d. 66 9688 9707 80 333640 540 559 CTTTGTACTTTCTTCATTTC n.d. 37 9690 9709 81 592752 543 562 TGTCTTTGTACTTTCTTCAT n.d. 63 9693 9712 712 592753 545 564 CCTGTCTTTGTACTTTCTTC n.d. 74 9695 9714 713 592754 547 566 TTCCTGTCTTTGTACTTTCT n.d. 72 9697 9716 714 592755 549 568 GTTTCCTGTCTTTGTACTTT n.d. 57 9699 9718 715 592756 568 587 AAGCCAAACGACTTCCAGCG 72 66 9718 9737 716 592757 570 589 ACAAGCCAAACGACTTCCAG 72 74 9720 9739 717 489516 572 591 CCACAAGCCAAACGACTTCC 85 82 9722 9741 718 592758 574 593 CACCACAAGCCAAACGACTT 72 73 9724 9743 719 592759 576 595 TACACCACAAGCCAAACGAC 74 68 9726 9745 720 592760 578 597 ATTACACCACAAGCCAAACG 67 61 9728 9747 721 592761 580 599 CAATTACACCACAAGCCAAA 64 56 9730 9749 722 150466 640 659 GATAACAGATGAGTTAAGGG 66 65 9790 9809 82 489521 642 661 AGGATAACAGATGAGTTAAG 79 78 9792 9811 723 592762 663 682 GGATACATTTCTACAGCTAG 91 87 9813 9832 724 592763 665 684 CAGGATACATTTCTACAGCT 92 89 9815 9834 725 592764 667 686 ATCAGGATACATTTCTACAG 88 83 9817 9836 726 592765 669 688 TTATCAGGATACATTTCTAC 77 72 9819 9838 727 592766 671 690 GTTTATCAGGATACATTTCT 90 89 9821 9840 728 592767 673 692 ATGTTTATCAGGATACATTT 82 76 9823 9842 729 592768 675 694 TAATGTTTATCAGGATACAT 80 79 9825 9844 730 592769 677 696 TTTAATGTTTATCAGGATAC 82 78 9827 9846 731 592770 679 698 TGTTTAATGTTTATCAGGAT 79 75 9829 9848 732 592771 681 700 AGTGTTTAATGTTTATCAGG 84 81 9831 9850 733 489526 692 711 TTTAAGATTACAGTGTTTAA 36 38 9842 9861 734 592772 694 713 CTTTTAAGATTACAGTGTTT 46 47 9844 9863 735 592773 696 715 CACTTTTAAGATTACAGTGT 39 42 9846 9865 736 592774 698 717 TACACTTTTAAGATTACAGT 21 24 9848 9867 737 592775 700 719 ATTACACTTTTAAGATTACA 3 0 9850 9869 738 489527 702 721 CAATTACACTTTTAAGATTA 0 0 9852 9871 739 150467 704 723 CACAATTACACTTTTAAGAT 58 73 9854 9873 83 592776 706 725 CACACAATTACACTTTTAAG 29 5 9856 9875 740 592777 708 727 GTCACACAATTACACTTTTA 59 49 9858 9877 741 592778 710 729 AAGTCACACAATTACACTTT 40 34 9860 9879 742 489528 712 731 AAAAGTCACACAATTACACT 31 27 9862 9881 743 592779 714 733 GAAAAAGTCACACAATTACA 21 7 9864 9883 744 592780 716 735 CTGAAAAAGTCACACAATTA 18 13 9866 9885 745 592781 718 737 CTCTGAAAAAGTCACACAAT 32 26 9868 9887 746 592782 720 739 AACTCTGAAAAAGTCACACA 35 20 9870 9889 747

TABLE 10  Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ ID ID SEQ SEQ NO: NO: % ID ID 1 1 inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO Site Site Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA 74 973 992 21 489529 722 741 GCAACTCTGAAAAAGTCACA 41 9872 9891 748 592783 724 743 AAGCAACTCTGAAAAAGTCA 34 9874 9893 749 592784 727 746 TTAAAGCAACTCTGAAAAAG 4 9877 9896 750 592785 729 748 CTTTAAAGCAACTCTGAAAA 36 9879 9898 751 592786 731 750 TACTTTAAAGCAACTCTGAA 28 9881 9900 752 592787 733 752 GGTACTTTAAAGCAACTCTG 48 9883 9902 753 592788 735 754 CAGGTACTTTAAAGCAACTC 38 9885 9904 754 592789 737 756 TACAGGTACTTTAAAGCAAC 20 9887 9906 755 592790 739 758 ACTACAGGTACTTTAAAGCA 26 9889 9908 756 592791 741 760 TCACTACAGGTACTTTAAAG 34 9891 9910 757 592792 743 762 TCTCACTACAGGTACTTTAA 50 9893 9912 758 592793 745 764 TTTCTCACTACAGGTACTTT 36 9895 9914 759 592794 747 766 AGTTTCTCACTACAGGTACT 53 9897 9916 760 592795 749 768 TCAGTTTCTCACTACAGGTA 37 9899 9918 761 150470 751 770 AATCAGTTTCTCACTACAGG 30 9901 9920 84 592796 753 772 TAAATCAGTTTCTCACTACA 21 9903 9922 762 150472 755 774 CATAAATCAGTTTCTCACTA 37 9905 9924 85 592797 757 776 ATCATAAATCAGTTTCTCAC 35 9907 9926 763 592798 759 778 TGATCATAAATCAGTTTCTC 35 9909 9928 764 592799 761 780 AGTGATCATAAATCAGTTTC 5 9911 9930 765 592800 763 782 CAAGTGATCATAAATCAGTT 21 9913 9932 766 592801 765 784 TCCAAGTGATCATAAATCAG 41 9915 9934 767 592802 767 786 CTTCCAAGTGATCATAAATC 44 9917 9936 768 592803 769 788 ATCTTCCAAGTGATCATAAA 30 9919 9938 769 592804 771 790 AAATCTTCCAAGTGATCATA 32 9921 9940 770 489534 792 811 CTGAGTTTTATAAAACTATA 4 9942 9961 771 150476 794 813 AACTGAGTTTTATAAAACTA 9 9944 9963 86 592805 796 815 TTAACTGAGTTTTATAAAAC 14 9946 9965 772 592806 798 817 TTTTAACTGAGTTTTATAAA 3 9948 9967 773 592807 800 819 CATTTTAACTGAGTTTTATA 13 9950 9969 774 489535 802 821 GACATTTTAACTGAGTTTTA 34 9952 9971 775 592808 804 823 CAGACATTTTAACTGAGTTT 40 9954 9973 776 592809 806 825 AACAGACATTTTAACTGAGT 36 9956 9975 777 592810 808 827 GAAACAGACATTTTAACTGA 25 9958 9977 778 592811 810 829 TTGAAACAGACATTTTAACT 24 9960 9979 779 592812 835 854 TTTAAGTCTGGCAAAATACA 23 9985 10004 780 592813 837 856 GATTTAAGTCTGGCAAAATA 31 9987 10006 781 592814 839 858 GTGATTTAAGTCTGGCAAAA 41 9989 10008 782 592815 841 860 CTGTGATTTAAGTCTGGCAA 49 9991 10010 783 592816 843 862 ATCTGTGATTTAAGTCTGGC 53 9993 10012 784 150481 845 864 CCATCTGTGATTTAAGTCTG 51 9995 10014 87 592817 847 866 ACCCATCTGTGATTTAAGTC 51 9997 10016 785 592818 849 868 ATACCCATCTGTGATTTAAG 43 9999 10018 786 592819 851 870 TAATACCCATCTGTGATTTA 42 10001 10020 787 592820 870 889 AAAGAAATTCTGACAAGTTT 22 10020 10039 788 489542 872 891 ACAAAGAAATTCTGACAAGT 13 10022 10041 789 592821 874 893 TGACAAAGAAATTCTGACAA 24 10024 10043 790 592822 876 895 AATGACAAAGAAATTCTGAC 25 10026 10045 791 592823 878 897 TGAATGACAAAGAAATTCTG 6 10028 10047 792 592824 880 899 CTTGAATGACAAAGAAATTC 24 10030 10049 793 489543 882 901 GGCTTGAATGACAAAGAAAT 29 10032 10051 794 592825 884 903 CAGGCTTGAATGACAAAGAA 35 10034 10053 795 592826 886 905 CACAGGCTTGAATGACAAAG 32 10036 10055 796 592827 888 907 TTCACAGGCTTGAATGACAA 41 10038 10057 797 592828 890 909 TATTCACAGGCTTGAATGAC 30 10040 10059 798 150492 909 928 AAGTGCCATACAGGGTTTTT 32 10059 10078 88 592829 911 930 ATAAGTGCCATACAGGGTTT 0 10061 10080 799 150493 913 932 TAATAAGTGCCATACAGGGT 24 10063 10082 89 592830 915 934 CATAATAAGTGCCATACAGG 26 10065 10084 800 150495 917 936 CTCATAATAAGTGCCATACA 35 10067 10086 90 150496 919 938 GCCTCATAATAAGTGCCATA 37 10069 10088 91 592831 921 940 TAGCCTCATAATAAGTGCCA 19 10071 10090 801 592832 923 942 AATAGCCTCATAATAAGTGC 0 10073 10092 802 592833 925 944 TTAATAGCCTCATAATAAGT 19 10075 10094 803 150497 927 946 TTTTAATAGCCTCATAATAA 17 10077 10096 92 592834 929 948 TCTTTTAATAGCCTCATAAT 27 10079 10098 804 592835 931 950 ATTCTTTTAATAGCCTCATA 27 10081 10100 805 150498 933 952 GGATTCTTTTAATAGCCTCA 39 10083 10102 93 592836 935 954 TTGGATTCTTTTAATAGCCT 24 10085 10104 806 592837 937 956 ATTTGGATTCTTTTAATAGC 0 10087 10106 807 592838 939 958 GAATTTGGATTCTTTTAATA 10 10089 10108 808 592839 941 960 TTGAATTTGGATTCTTTTAA 13 10091 10110 809 592840 943 962 GTTTGAATTTGGATTCTTTT 29 10093 10112 810 592841 945 964 TAGTTTGAATTTGGATTCTT 31 10095 10114 811 592842 947 966 TTTAGTTTGAATTTGGATTC 8 10097 10116 812 592843 949 968 TTTTTAGTTTGAATTTGGAT 10 n/a n/a 813 592844 951 970 TTTTTTTAGTTTGAATTTGG 7 n/a n/a 814

Example 2: Inhibition of Human SOD-1 in HepG2 Cells by MOE Gapmers

Modified oligonucleotides were designed targeting asuperoxide dismutase 1, soluble (SOD-1) nucleic acid and were tested for their effects on SOD-1 mRNA in vitro. ISIS 146143, ISIS150438-150440, ISIS 150442, ISIS 150450, ISIS 150455-150457, ISIS 150459, ISIS 150461, ISIS 150469, ISIS 150473, ISIS 150478, ISIS 150484, ISIS 150486, ISIS 150494, ISIS 150508-150510, ISIS 333607, ISIS 333608, ISIS 333611, ISIS 333618, previously disclosed in WO 2005/040180, were also included in this assay. The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 5,000 nM modified oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR.

Human primer probe set RTS3898 was used to measure mRNA levels. In cases where the oligonucleotide overlapped the amplicon of the primer probe set, an alternative primer probe set, HTS90, was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of SOD-1, relative to untreated control cells. ‘n.d.’ indicates that inhibition levels were not measured using the particular primer probe set.

The newly designed modified oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment is comprised of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ directions comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human SOD-1 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the human SOD-1 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000 to 18704000). ‘n/a’ indicates that the modified oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 11  Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ ID ID SEQ SEQ NO: NO: % ID ID 1 1 inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO Site Site Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA 64 973 992 21 596301 550 569 CGTTTCCTGTCTTTGTACTT n.d. 9700 9719 815 596302 569 588 CAAGCCAAACGACTTCCAGC 54 9719 9738 816 596303 571 590 CACAAGCCAAACGACTTCCA 47 9721 9740 817 596304 573 592 ACCACAAGCCAAACGACTTC 28 9723 9742 818 596305 575 594 ACACCACAAGCCAAACGACT 49 9725 9744 819 596306 577 596 TTACACCACAAGCCAAACGA 24 9727 9746 820 596307 641 660 GGATAACAGATGAGTTAAGG 48 9791 9810 821 596308 664 683 AGGATACATTTCTACAGCTA 79 9814 9833 822 596309 666 685 TCAGGATACATTTCTACAGC 70 9816 9835 823 596310 668 687 TATCAGGATACATTTCTACA 58 9818 9837 824 489524 672 691 TGTTTATCAGGATACATTTC 52 9822 9841 825 596311 674 693 AATGTTTATCAGGATACATT 54 9824 9843 826 596312 676 695 TTAATGTTTATCAGGATACA 34 9826 9845 827 596313 678 697 GTTTAATGTTTATCAGGATA 71 9828 9847 828 596314 680 699 GTGTTTAATGTTTATCAGGA 73 9830 9849 829 596315 693 712 TTTTAAGATTACAGTGTTTA 13 9843 9862 830 596316 695 714 ACTTTTAAGATTACAGTGTT 24 9845 9864 831 596317 697 716 ACACTTTTAAGATTACAGTG 15 9847 9866 832 596318 699 718 TTACACTTTTAAGATTACAG 0 9849 9868 833 596319 701 720 AATTACACTTTTAAGATTAC 1 9851 9870 834 596320 705 724 ACACAATTACACTTTTAAGA 0 9855 9874 835 596321 707 726 TCACACAATTACACTTTTAA 15 9857 9876 836 596322 711 730 AAAGTCACACAATTACACTT 15 9861 9880 837 596323 715 734 TGAAAAAGTCACACAATTAC 0 9865 9884 838 596324 717 736 TCTGAAAAAGTCACACAATT 5 9867 9886 839 596325 719 738 ACTCTGAAAAAGTCACACAA 21 9869 9888 840 596326 723 742 AGCAACTCTGAAAAAGTCAC 14 9873 9892 841 596327 730 749 ACTTTAAAGCAACTCTGAAA 0 9880 9899 842 489530 732 751 GTACTTTAAAGCAACTCTGA 22 9882 9901 843 596328 734 753 AGGTACTTTAAAGCAACTCT 36 9884 9903 844 596329 740 759 CACTACAGGTACTTTAAAGC 18 9890 9909 845 150469 742 761 CTCACTACAGGTACTTTAAA 25 9892 9911 94 596330 744 763 TTCTCACTACAGGTACTTTA 28 9894 9913 846 596331 746 765 GTTTCTCACTACAGGTACTT 30 9896 9915 847 596332 748 767 CAGTTTCTCACTACAGGTAC 25 9898 9917 848 596333 750 769 ATCAGTTTCTCACTACAGGT 22 9900 9919 849 489531 752 771 AAATCAGTTTCTCACTACAG 0 9902 9921 850 596334 756 775 TCATAAATCAGTTTCTCACT 21 9906 9925 851 596335 760 779 GTGATCATAAATCAGTTTCT 37 9910 9929 852 489532 762 781 AAGTGATCATAAATCAGTTT 8 9912 9931 853 596336 764 783 CCAAGTGATCATAAATCAGT 39 9914 9933 854 436935 766 785 TTCCAAGTGATCATAAATCA 18 9916 9935 855 596337 768 787 TCTTCCAAGTGATCATAAAT 12 9918 9937 856 150473 770 789 AATCTTCCAAGTGATCATAA 4 9920 9939 95 596338 795 814 TAACTGAGTTTTATAAAACT 0 9945 9964 857 596339 807 826 AAACAGACATTTTAACTGAG 4 9957 9976 858 596340 809 828 TGAAACAGACATTTTAACTG 0 9959 9978 859 150478 811 830 ATTGAAACAGACATTTTAAC 0 9961 9980 96 596341 836 855 ATTTAAGTCTGGCAAAATAC 16 9986 10005 860 596342 840 859 TGTGATTTAAGTCTGGCAAA 34 9990 10009 861 489539 842 861 TCTGTGATTTAAGTCTGGCA 44 9992 10011 862 596343 844 863 CATCTGTGATTTAAGTCTGG 29 9994 10013 863 596344 846 865 CCCATCTGTGATTTAAGTCT 41 9996 10015 864 596345 848 867 TACCCATCTGTGATTTAAGT 50 9998 10017 865 596346 850 869 AATACCCATCTGTGATTTAA 0 10000 10019 866 489540 852 871 TTAATACCCATCTGTGATTT 11 10002 10021 867 150484 871 890 CAAAGAAATTCTGACAAGTT 7 10021 10040 97 596347 873 892 GACAAAGAAATTCTGACAAG 8 10023 10042 868 596348 877 896 GAATGACAAAGAAATTCTGA 0 10027 10046 869 596349 883 902 AGGCTTGAATGACAAAGAAA 27 10033 10052 870 150486 885 904 ACAGGCTTGAATGACAAAGA 19 10035 10054 98 596350 910 929 TAAGTGCCATACAGGGTTTT 13 10060 10079 871 596351 914 933 ATAATAAGTGCCATACAGGG 18 10064 10083 872 150494 916 935 TCATAATAAGTGCCATACAG 0 10066 10085 99 596352 918 937 CCTCATAATAAGTGCCATAC 23 10068 10087 873 596353 920 939 AGCCTCATAATAAGTGCCAT 6 10070 10089 874 596354 922 941 ATAGCCTCATAATAAGTGCC 19 10072 10091 875 596355 928 947 CTTTTAATAGCCTCATAATA 0 10078 10097 876 596356 930 949 TTCTTTTAATAGCCTCATAA 5 10080 10099 877 596357 932 951 GATTCTTTTAATAGCCTCAT 4 10082 10101 878 596358 934 953 TGGATTCTTTTAATAGCCTC 13 10084 10103 879 596359 936 955 TTTGGATTCTTTTAATAGCC 14 10086 10105 880 596360 938 957 AATTTGGATTCTTTTAATAG 14 10088 10107 881 596361 940 959 TGAATTTGGATTCTTTTAAT 0 10090 10109 882 596362 946 965 TTAGTTTGAATTTGGATTCT 0 10096 10115 883 596363 948 967 TTTTAGTTTGAATTTGGATT 0 n/a n/a 884 596364 950 969 TTTTTTAGTTTGAATTTGGA 0 n/a n/a 885

TABLE 12  Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: % % NO: NO: 1 1 inhibition inhibition 2 2 SEQ ISIS Start Stop with with Start Stop ID NO Site Site Sequence RTS3898 HTS90 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA 66 n.d. 973 992 21 596230 246 265 CAGTCCTTTAATGCTTCCCC 51 40 5000 5019 886 596231 248 267 GTCAGTCCTTTAATGCTTCC 34 34 5002 5021 887 596232 250 269 CAGTCAGTCCTTTAATGCTT 35 29 5004 5023 888 596233 252 271 TTCAGTCAGTCCTTTAATGC 24 21 5006 5025 889 596234 256 275 GGCCTTCAGTCAGTCCTTTA 41 39 5010 5029 890 150450 258 277 CAGGCCTTCAGTCAGTCCTT 56 51 5012 5031 100 596235 260 279 TGCAGGCCTTCAGTCAGTCC 42 46 5014 5033 891 596236 262 281 CATGCAGGCCTTCAGTCAGT 37 33 5016 5035 892 596237 278 297 TCATGAACATGGAATCCATG 24 19 5032 5051 893 596238 280 299 ACTCATGAACATGGAATCCA 27 20 5034 5053 894 596239 295 314 CTGTATTATCTCCAAACTCA 32 28 5049 5068 895 596240 309 328 ACTGGTACAGCCTGCTGTAT 22 28 n/a n/a 896 596241 311 330 GCACTGGTACAGCCTGCTGT 31 24 n/a n/a 897 596242 313 332 CTGCACTGGTACAGCCTGCT 38 29 n/a n/a 898 596243 315 334 ACCTGCACTGGTACAGCCTG 46 48 n/a n/a 899 596244 341 360 TTTCTGGATAGAGGATTAAA 6 14 7657 7676 900 596245 343 362 GTTTTCTGGATAGAGGATTA 28 39 7659 7678 901 596246 347 366 CCGTGTTTTCTGGATAGAGG 44 37 7663 7682 902 596247 349 368 CACCGTGTTTTCTGGATAGA 24 11 7665 7684 903 596248 351 370 CCCACCGTGTTTTCTGGATA 46 40 7667 7686 904 596249 353 372 GGCCCACCGTGTTTTCTGGA 46 41 7669 7688 905 596250 355 374 TTGGCCCACCGTGTTTTCTG 35 26 7671 7690 906 596251 357 376 CTTTGGCCCACCGTGTTTTC 31 15 7673 7692 907 596252 359 378 TCCTTTGGCCCACCGTGTTT 30 23 7675 7694 908 596253 383 402 AAGTCTCCAACATGCCTCTC 20 6 n/a n/a 909 596254 387 406 GCCCAAGTCTCCAACATGCC 61 53 8442 8461 910 596255 389 408 TTGCCCAAGTCTCCAACATG 41 33 8444 8463 911 596256 391 410 CATTGCCCAAGTCTCCAACA 39 25 8446 8465 912 150455 393 412 CACATTGCCCAAGTCTCCAA 36 19 8448 8467 101 596257 397 416 CAGTCACATTGCCCAAGTCT 40 27 8452 8471 913 596258 401 420 TCAGCAGTCACATTGCCCAA 52 42 8456 8475 914 596259 403 422 TGTCAGCAGTCACATTGCCC 55 49 8458 8477 915 596260 405 424 TTTGTCAGCAGTCACATTGC 26 16 8460 8479 916 596261 407 426 TCTTTGTCAGCAGTCACATT 20 11 8462 8481 917 596262 409 428 CATCTTTGTCAGCAGTCACA 34 13 8464 8483 918 596263 411 430 ACCATCTTTGTCAGCAGTCA 41 30 8466 8485 919 596264 415 434 CCACACCATCTTTGTCAGCA 39 20 8470 8489 920 596265 417 436 GGCCACACCATCTTTGTCAG 23 5 8472 8491 921 150456 419 438 TCGGCCACACCATCTTTGTC 32 28 8474 8493 102 150457 421 440 CATCGGCCACACCATCTTTG 34 38 8476 8495 103 596266 423 442 CACATCGGCCACACCATCTT 27 13 8478 8497 922 596267 425 444 GACACATCGGCCACACCATC 45 30 8480 8499 923 150459 427 446 TAGACACATCGGCCACACCA 46 36 8482 8501 104 596268 429 448 AATAGACACATCGGCCACAC 30 25 8484 8503 924 596269 431 450 TCAATAGACACATCGGCCAC 35 0 8486 8505 925 596270 433 452 CTTCAATAGACACATCGGCC 39 16 8488 8507 926 596271 435 454 ATCTTCAATAGACACATCGG 16 0 8490 8509 927 596272 437 456 GAATCTTCAATAGACACATC 22 11 8492 8511 928 596273 439 458 CAGAATCTTCAATAGACACA 17 0 8494 8513 929 596274 441 460 CACAGAATCTTCAATAGACA 10 14 8496 8515 930 596275 443 462 ATCACAGAATCTTCAATAGA 11 10 8498 8517 931 596276 445 464 AGATCACAGAATCTTCAATA 14 29 8500 8519 932 596277 447 466 TGAGATCACAGAATCTTCAA n.d. 30 8502 8521 933 596278 449 468 AGTGAGATCACAGAATCTTC n.d. 30 8504 8523 934 596279 453 472 TGAGAGTGAGATCACAGAAT n.d. 18 8508 8527 935 333618 457 476 CTCCTGAGAGTGAGATCACA n.d. 27 8512 8531 105 596281 459 478 GTCTCCTGAGAGTGAGATCA n.d. 23 8514 8533 936 596282 461 480 TGGTCTCCTGAGAGTGAGAT n.d. 24 8516 8535 937 596283 463 482 AATGGTCTCCTGAGAGTGAG n.d. 22 8518 8537 938 596284 465 484 GCAATGGTCTCCTGAGAGTG n.d. 57 8520 8539 939 596285 467 486 ATGCAATGGTCTCCTGAGAG n.d. 0 8522 8541 940 596286 469 488 TGATGCAATGGTCTCCTGAG n.d. 1 8524 8543 941 596287 471 490 AATGATGCAATGGTCTCCTG n.d. 0 8526 8545 942 596288 473 492 CCAATGATGCAATGGTCTCC n.d. 8 8528 8547 943 596289 475 494 GGCCAATGATGCAATGGTCT n.d. 9 8530 8549 944 596290 477 496 GCGGCCAATGATGCAATGGT n.d. 13 8532 8551 945 596291 479 498 GTGCGGCCAATGATGCAATG n.d. 12 8534 8553 946 596292 481 500 GTGTGCGGCCAATGATGCAA n.d. 15 8536 8555 947 596293 483 502 CAGTGTGCGGCCAATGATGC n.d. 0 8538 8557 948 596294 485 504 ACCAGTGTGCGGCCAATGAT n.d. 0 8540 8559 949 596295 487 506 CCACCAGTGTGCGGCCAATG n.d. 22 8542 8561 950 596296 489 508 GACCACCAGTGTGCGGCCAA n.d. 16 n/a n/a 951 596297 491 510 TGGACCACCAGTGTGCGGCC n.d. 28 n/a n/a 952 150461 493 512 CATGGACCACCAGTGTGCGG n.d. 25 n/a n/a 106 596298 495 514 TTCATGGACCACCAGTGTGC n.d. 21 n/a n/a 953 596299 497 516 TTTTCATGGACCACCAGTGT n.d. 17 n/a n/a 954 596300 499 518 CTTTTTCATGGACCACCAGT n.d. 9 n/a n/a 955

Example 3: Inhibition of Human SOD-1 in HepG2 Cells by Deoxy, MOE and cEt Gapmers

Modified oligonucleotides were designed targeting a superoxide dismutase 1, soluble (SOD-1) nucleic acid and were tested for their effects on SOD-1 mRNA in vitro. ISIS 333611, which was previously described in WO 2005/040180, was included as a benchmark. ISIS 590067, ISIS 590074, ISIS 590082, ISIS 590130, ISIS 590138, and ISIS 590146, which are 5-10-5 MOE gapmers as described above in Example 1, were also included in this assay. ISIS 590512, which has a similar sequence as ISIS 333611 but with deoxy, MOE, and cEt sugar modifications, was also included in this study.

The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 3,000 nM modified oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR.

Human primer probe set RTS3898 was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells. ‘n.d.’ indicates that inhibition levels were not measured.

The newly designed modified oligonucleotides in the Tables below were designed as deoxy, MOE, and cEt gapmers. The gapmers are 17 nucleosides in length wherein each nucleoside has a MOE sugar modification, a cEt sugar modification, or a deoxy moiety. The sugar chemistry of each oligonucleotide is denoted as in the Chemistry column, where ‘k’ indicates a cEt modified sugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’ indicates a 2′-MOE modified sugar. The internucleoside linkages throughout each gapmer are phosphorothioate linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human SOD-1 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the human SOD-1 genomic sequence, designated herein as SEQ ID NO: (GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000 to 18704000). ‘n/a’ indicates that the modified oligonucleotide does not target that particular gene sequence with S100 complementarity.

TABLE 13  Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: NO: 1 1 % 2 2 SEQ ISIS Start Stop inhi- Start Stop NO NO Site Site Sequence Chemistry bition Site Site ID 590434 1 17 CCACTCTGGCCC eeekkdddddddkkeee 17 807 823 956 CAAAC 590435 2 18 CCCACTCTGGCC eeekkdddddddkkeee 15 808 824 957 CCAAA 590436 3 19 GCCCACTCTGGC eeekkdddddddkkeee 22 809 825 958 CCCAA 590437 4 20 CGCCCACTCTGG eeekkdddddddkkeee 14 810 826 959 CCCCA 590438 35 51 CGACTACTTTAT eeekkdddddddkkeee 12 841 857 960 AGGCC 590439 36 52 GCGACTACTTTA eeekkdddddddkkeee 12 842 858 961 TAGGC 590440 37 53 CGCGACTACTTT eeekkdddddddkkeee 11 843 859 962 ATAGG 590441 38 54 CCGCGACTACTT eeekkdddddddkkeee 5 844 860 963 TATAG 590442 76 92 CGCTGCAGGAGA eeekkdddddddkkeee 0 882 898 964 CTACG 590443 77 93 ACGCTGCAGGAG eeekkdddddddkkeee 25 883 899 965 ACTAC 590444 167 183 TCGCCCTTCAGC eeekkdddddddkkeee 31 973 989 966 ACGCA 590445 168 184 GTCGCCCTTCAG eeekkdddddddkkeee 28 974 990 967 CACGC 590512 169 185 CGTCGCCCTTCA eeekkdddddddkkeee 8 975 991 968 GCACG 590446 170 186 CCGTCGCCCTTC eeekkdddddddkkeee 27 976 992 969 AGCAC 590447 171 187 GCCGTCGCCCTT eeekkdddddddkkeee 33 977 993 970 CAGCA 590448 202 218 TCTGCTCGAAAT eeekkdddddddkkeee 34 1008 1024 971 TGATG 590449 203 219 TTCTGCTCGAAA eeekkdddddddkkeee 18 1009 1025 972 TTGAT 590450 204 220 CTTCTGCTCGAA eeekkdddddddkkeee 13 1010 1026 973 ATTGA 590451 205 221 CCTTCTGCTCGA eeekkdddddddkkeee 16 1011 1027 974 AATTG 590452 206 222 TCCTTCTGCTCG eeekkdddddddkkeee 14 n/a n/a 975 AAATT 590453 207 223 TTCCTTCTGCTCG eeekkdddddddkkeee 13 n/a n/a 976 AAAT 590454 208 224 TTTCCTTCTGCTC eeekkdddddddkkeee 6 n/a n/a 977 GAAA 590455 209 225 CTTTCCTTCTGCT eeekkdddddddkkeee 0 n/a n/a 978 CGAA 590456 210 226 ACTTTCCTTCTGC eeekkdddddddkkeee 0 n/a n/a 979 TCGA 590457 211 227 TACTTTCCTTCTG eeekkdddddddkkeee n.d. n/a n/a 980 CTCG 590458 212 228 TTACTTTCCTTCT eeekkdddddddkkeee n.d. n/a n/a 981 GCTC 590459 213 229 ATTACTTTCCTTC eeekkdddddddkkeee n.d. n/a n/a 982 TGCT 590461 214 230 CATTACTTTCCTT eeekkdddddddkkeee n.d. n/a n/a 983 CTGC 590462 215 231 CCATTACTTTCCT eeekkdddddddkkeee n.d. n/a n/a 984 TCTG 590463 216 232 TCCATTACTTTCC eeekkdddddddkkeee n.d. n/a n/a 985 TTCT 590464 217 233 GTCCATTACTTTC eeekkdddddddkkeee n.d. n/a n/a 986 CTTC 590465 218 234 GGTCCATTACTT eeekkdddddddkkeee n.d. 4972 4988 987 TCCTT 590466 219 235 TGGTCCATTACT eeekkdddddddkkeee 5 4973 4989 988 TTCCT 590467 220 236 CTGGTCCATTAC eeekkdddddddkkeee 11 4974 4990 989 TTTCC 590468 221 237 ACTGGTCCATTA eeekkdddddddkkeee 14 4975 4991 990 CTTTC 590469 222 238 CACTGGTCCATT eeekkdddddddkkeee 12 4976 4992 991 ACTTT 590470 223 239 TCACTGGTCCAT eeekkdddddddkkeee 15 4977 4993 992 TACTT 590471 224 240 TTCACTGGTCCA eeekkdddddddkkeee 14 4978 4994 993 TTACT 590472 225 241 CTTCACTGGTCC eeekkdddddddkkeee 11 4979 4995 994 ATTAC 590473 226 242 CCTTCACTGGTC eeekkdddddddkkeee 8 4980 4996 995 CATTA 590474 227 243 ACCTTCACTGGT eeekkdddddddkkeee 44 4981 4997 996 CCATT 590475 228 244 CACCTTCACTGG eeekkdddddddkkeee 53 4982 4998 997 TCCAT 590476 229 245 ACACCTTCACTG eeekkdddddddkkeee 20 4983 4999 998 GTCCA 590477 230 246 CACACCTTCACT eeekkdddddddkkeee 12 4984 5000 999 GGTCC 590478 231 247 CCACACCTTCAC eeekkdddddddkkeee 36 4985 5001 1000 TGGTC 590479 232 248 CCCACACCTTCA eeekkdddddddkkeee 18 4986 5002 1001 CTGGT 590480 233 249 CCCCACACCTTC eeekkdddddddkkeee 14 4987 5003 1002 ACTGG 590481 235 251 TTCCCCACACCT eeekkdddddddkkeee 8 4989 5005 1003 TCACT 590482 236 252 CTTCCCCACACC eeekkdddddddkkeee 29 4990 5006 1004 TTCAC 590483 237 253 GCTTCCCCACAC eeekkdddddddkkeee 36 4991 5007 1005 CTTCA 590484 238 254 TGCTTCCCCACA eeekkdddddddkkeee 43 4992 5008 1006 CCTTC 590485 239 255 ATGCTTCCCCAC eeekkdddddddkkeee 41 4993 5009 1007 ACCTT 590486 240 256 AATGCTTCCCCA eeekkdddddddkkeee 35 4994 5010 1008 CACCT 590487 241 257 TAATGCTTCCCC eeekkdddddddkkeee 52 4995 5011 1009 ACACC 590488 264 280 ATGCAGGCCTTC eeekkdddddddkkeee 37 5018 5034 1010 AGTCA 590489 265 281 CATGCAGGCCTT eeekkdddddddkkeee 41 5019 5035 1011 CAGTC 590490 266 282 CCATGCAGGCCT eeekkdddddddkkeee 21 5020 5036 1012 TCAGT 590491 267 283 TCCATGCAGGCC eeekkdddddddkkeee 18 5021 5037 1013 TTCAG 590492 268 284 ATCCATGCAGGC eeekkdddddddkkeee 27 5022 5038 1014 CTTCA 590493 269 285 AATCCATGCAGG eeekkdddddddkkeee 13 5023 5039 1015 CCTTC 590494 270 286 GAATCCATGCAG eeekkdddddddkkeee 9 5024 5040 1016 GCCTT 590495 271 287 GGAATCCATGCA eeekkdddddddkkeee 7 5025 5041 1017 GGCCT 590496 272 288 TGGAATCCATGC eeekkdddddddkkeee 12 5026 5042 1018 AGGCC 590497 273 289 ATGGAATCCATG eeekkdddddddkkeee 9 5027 5043 1019 CAGGC 590498 274 290 CATGGAATCCAT eeekkdddddddkkeee 14 5028 5044 1020 GCAGG 590499 275 291 ACATGGAATCCA eeekkdddddddkkeee 0 5029 5045 1021 TGCAG 590500 276 292 AACATGGAATCC eeekkdddddddkkeee 10 5030 5046 1022 ATGCA 590501 277 293 GAACATGGAATC eeekkdddddddkkeee 9 5031 5047 1023 CATGC 590502 278 294 TGAACATGGAAT eeekkdddddddkkeee 2 5032 5048 1024 CCATG 590503 279 295 ATGAACATGGAA eeekkdddddddkkeee 8 5033 5049 1025 TCCAT 590504 316 332 CTGCACTGGTAC eeekkdddddddkkeee 3 7632 7648 1026 AGCCT 590505 317 333 CCTGCACTGGTA eeekkdddddddkkeee 17 7633 7649 1027 CAGCC 590506 318 334 ACCTGCACTGGT eeekkdddddddkkeee 12 7634 7650 1028 ACAGC 590507 319 335 GACCTGCACTGG eeekkdddddddkkeee 7 7635 7651 1029 TACAG 590508 320 336 GGACCTGCACTG eeekkdddddddkkeee 7 7636 7652 1030 GTACA 590509 321 337 AGGACCTGCACT eeekkdddddddkkeee 4 7637 7653 1031 GGTAC 590510 322 338 GAGGACCTGCAC eeekkdddddddkkeee 17 7638 7654 1032 TGGTA 590511 323 339 TGAGGACCTGCA eeekkdddddddkkeee 8 7639 7655 1033 CTGGT

TABLE 14 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmers targeting SEQ ID NO: 1 and/or 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence Chemistry inhibition Site Site NO 590512 169 185 CGTCGCCCTTCA eeekkdddddddkkeee 45  975  991  968 GCACG 590513 324 340 GTGAGGACCTG eeekkdddddddkkeee 21 7640 7656 1034 CACTGG 590514 325 341 AGTGAGGACCT eeekkdddddddkkeee 21 7641 7657 1035 GCACTG 590515 326 342 AAGTGAGGACC eeekkdddddddkkeee 16 7642 7658 1036 TGCACT 590516 327 343 AAAGTGAGGAC eeekkdddddddkkeee 20 7643 7659 1037 CTGCAC 590517 328 344 TAAAGTGAGGA eeekkdddddddkkeee 19 7644 7660 1038 CCTGCA 590518 329 345 TTAAAGTGAGG eeekkdddddddkkeee 14 7645 7661 1039 ACCTGC 590519 330 346 ATTAAAGTGAG eeekkdddddddkkeee Si 7646 7662 1040 GACCTG 590520 331 347 GATTAAAGTGA eeekkdddddddkkeee 8 7647 7663 1041 GGACCT 590521 332 348 GGATTAAAGTG eeekkdddddddkkeee 30 7648 7664 1042 AGGACC 590522 333 349 AGGATTAAAGT eeekkdddddddkkeee 23 7649 7665 1043 GAGGAC 590523 334 350 GAGGATTAAAG eeekkdddddddkkeee 40 7650 7666 1044 TGAGGA 590524 335 351 AGAGGATTAAA eeekkdddddddkkeee 16 7651 7667 1045 GTGAGG 590525 336 352 TAGAGGATTAA eeekkdddddddkkeee 21 7652 7668 1046 AGTGAG 590526 337 353 ATAGAGGATTA eeekkdddddddkkeee  9 7653 7669 1047 AAGTGA 590527 338 354 GATAGAGGATT eeekkdddddddkkeee  8 7654 7670 1048 AAAGTG 590528 339 355 GGATAGAGGAT eeekkdddddddkkeee 14 7655 7671 1049 TAAAGT 590530 340 356 TGGATAGAGGA eeekkdddddddkkeee 23 7656 7672 1050 TTAAAG 590531 341 357 CTGGATAGAGG eeekkdddddddkkeee 26 7657 7673 1051 ATTAAA 590532 342 358 TCTGGATAGAG eeekkdddddddkkeee 25 7658 7674 1052 GATTAA 590533 360 376 CTTTGGCCCACC eeekkdddddddkkeee 41 7676 7692 1053 GTGTT 590534 361 377 CCTTTGGCCCAC eeekkdddddddkkeee 46 7677 7693 1054 CGTGT 590535 362 378 TCCTTTGGCCCA eeekkdddddddkkeee 39 7678 7694 1055 CCGTG 590536 363 379 ATCCTTTGGCCC eeekkdddddddkkeee n.d. 7679 7695 1056 ACCGT 590537 364 380 CATCCTTTGGCC eeekkdddddddkkeee n.d. 7680 7696 1057 CACCG 590538 365 381 TCATCCTTTGGC eeekkdddddddkkeee n.d. 7681 7697 1058 CCACC 590539 366 382 TTCATCCTTTGG eeekkdddddddkkeee n.d. 7682 7698 1059 CCCAC 590540 367 383 CTTCATCCTTTG eeekkdddddddkkeee n.d. 7683 7699 1060 GCCCA 590541 368 384 TCTTCATCCTTT eeekkdddddddkkeee n.d. 7684 7700 1061 GGCCC 590542 369 385 CTCTTCATCCTT eeekkdddddddkkeee n.d. 7685 7701 1062 TGGCC 590543 370 386 TCTCTTCATCCT eeekkdddddddkkeee n.d. 7686 7702 1063 TTGGC 590544 371 387 CTCTCTTCATCC eeekkdddddddkkeee  2 7687 7703 1064 TTTGG 590545 374 390 TGCCTCTCTTCA eeekkdddddddkkeee  6 n/a n/a 1065 TCCTT 590546 375 391 ATGCCTCTCTTC eeekkdddddddkkeee  0 n/a n/a 1066 ATCCT 590547 376 392 CATGCCTCTCTT eeekkdddddddkkeee 14 n/a n/a 1067 CATCC 590548 377 393 ACATGCCTCTCT eeekkdddddddkkeee  0 n/a n/a 1068 TCATC 590549 378 394 AACATGCCTCTC eeekkdddddddkkeee 13 n/a n/a 1069 TTCAT 590550 379 395 CAACATGCCTCT eeekkdddddddkkeee  3 n/a n/a 1070 CTTCA 590551 380 396 CCAACATGCCTC eeekkdddddddkkeee  0 n/a n/a 1071 TCTTC 590552 381 397 TCCAACATGCCT eeekkdddddddkkeee  0 n/a n/a 1072 CTCTT 590553 382 398 CTCCAACATGCC eeekkdddddddkkeee  5 n/a n/a 1073 TCTCT 590554 383 399 TCTCCAACATGC eeekkdddddddkkeee 10 n/a n/a 1074 CTCTC 590555 384 400 GTCTCCAACATG eeekkdddddddkkeee  8 n/a n/a 1075 CCTCT 590556 402 418 AGCAGTCACATT eeekkdddddddkkeee 18 8457 8473 1076 GCCCA 590557 403 419 CAGCAGTCACA eeekkdddddddkkeee  7 8458 8474 1077 TTGCCC 590558 429 445 AGACACATCGG eeekkdddddddkkeee 21 8484 8500 1078 CCACAC 590559 436 452 CTTCAATAGACA eeekkdddddddkkeee  9 8491 8507 1079 CATCG  590560 449 465 GAGATCACAGA eeekkdddddddkkeee 13 8504 8520 1080 ATCTTC 590561 501 517 TTTTTCATGGAC eeekkdddddddkkeee 76 n/a n/a 1081 CACCA 590562 502 518 CTTTTTCATGGA eeekkdddddddkkeee 87 n/a n/a 1082 CCACC 590563 503 519 GCTTTTTCATGG eeekkdddddddkkeee 71 n/a n/a 1083 ACCAC 590564 504 520 TGCTTTTTCATG eeekkdddddddkkeee 51 n/a n/a 1084 GACCA 590565 505 521 CTGCTTTTTCAT eeekkdddddddkkeee 65 9655 9671 1085 GGACC 590566 506 522 TCTGCTTTTTCA eeekkdddddddkkeee 55 9656 9672 1086 TGGAC 590567 507 523 ATCTGCTTTTTC eeekkdddddddkkeee 42 9657 9673 1087 ATGGA 590568 508 524 CATCTGCTTTTT eeekkdddddddkkeee 70 9658 9674 1088 CATGG 590569 509 525 TCATCTGCTTTT eeekkdddddddkkeee 71 9659 9675 1089 TCATG 590570 510 526 GTCATCTGCTTT eeekkdddddddkkeee 74 9660 9676 1090 TTCAT 590571 511 527 AGTCATCTGCTT eeekkdddddddkkeee 76 9661 9677 1091 TTTCA 590572 512 528 AAGTCATCTGCT eeekkdddddddkkeee 83 9662 9678 1092 TTTTC 590573 513 529 CAAGTCATCTGC eeekkdddddddkkeee 42 9663 9679 1093 TTTTT 590574 514 530 CCAAGTCATCTG eeekkdddddddkkeee 50 9664 9680 1094 CTTTT 590575 515 531 CCCAAGTCATCT eeekkdddddddkkeee 72 9665 9681 1095 GCTTT 590576 516 532 GCCCAAGTCATC eeekkdddddddkkeee 93 9666 9682 1096 TGCTT 590577 517 533 TGCCCAAGTCAT eeekkdddddddkkeee 90 9667 9683 1097 CTGCT 590578 518 534 TTGCCCAAGTCA eeekkdddddddkkeee 92 9668 9684 1098 TCTGC 590579 524 540 CCACCTTTGCCC eeekkdddddddkkeee 91 9674 9690 1099 AAGTC 590580 525 541 TCCACCTTTGCC eeekkdddddddkkeee 88 9675 9691 1100 CAAGT 590581 526 542 TTCCACCTTTGC eeekkdddddddkkeee 87 9676 9692 1101 CCAAG 590582 527 543 TTTCCACCTTTG eeekkdddddddkkeee 78 9677 9693 1102 CCCAA 590583 528 544 ATTTCCACCTTT eeekkdddddddkkeee 63 9678 9694 1103 GCCCA 590584 529 545 CATTTCCACCTT eeekkdddddddkkeee 73 9679 9695 1104 TGCCC 590585 530 546 TCATTTCCACCT eeekkdddddddkkeee 57 9680 9696 1105 TTGCC 590586 531 547 TTCATTTCCACC eeekkdddddddkkeee 33 9681 9697 1106 TTTGC 590587 533 549 TCTTCATTTCCA eeekkdddddddkkeee 31 9683 9699 1107 CCTTT 590588 536 552 CTTTCTTCATTT eeekkdddddddkkeee 11 9686 9702 1108 CCACC 590589 537 553 ACTTTCTTCATT eeekkdddddddkkeee 15 9687 9703 1109 TCCAC 590590 538 554 TACTTTCTTCAT eeekkdddddddkkeee 18 9688 9704 1110 TTCCA

TABLE 15 Percent inhibition of SOD-1 mRNA by deoxy MOE and cEt gapmers targeting SEQ ID NO: 1 and/or 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence Chemistry inhibition Site Site NO 590512 169 185 CGTCGCCCTTCAG eeekkdddddddkkeee 21  975  991  968 CACG 590591 582 598 AATTACACCACA eeekkdddddddkkeee 21 9732 9748 1111 AGCCA 590592 583 599 CAATTACACCAC eeekkdddddddkkeee 33 9733 9749 1112 AAGCC 590593 584 600 CCAATTACACCA eeekkdddddddkkeee 29 9734 9750 1113 CAAGC 590594 585 601 CCCAATTACACC eeekkdddddddkkeee 29 9735 9751 1114 ACAAG 590595 588 604 GATCCCAATTAC eeekkdddddddkkeee  3 9738 9754 1115 ACCAC 590596 589 605 CGATCCCAATTAC eeekkdddddddkkeee 12 9739 9755 1116 ACCA 590597 590 606 GCGATCCCAATT eeekkdddddddkkeee 19 9740 9756 1117 ACACC 590598 591 607 GGCGATCCCAAT eeekkdddddddkkeee  9 9741 9757 1118 TACAC 590599 592 608 GGGCGATCCCAA eeekkdddddddkkeee 18 9742 9758 1119 TTACA 590600 593 609 TGGGCGATCCCA eeekkdddddddkkeee 20 9743 9759 1120 ATTAC 590601 594 610 TTGGGCGATCCC eeekkdddddddkkeee 26 9744 9760 1121 AATTA 590602 595 611 ATTGGGCGATCC eeekkdddddddkkeee 19 9745 9761 1122 CAATT 590603 596 612 TATTGGGCGATCC eeekkdddddddkkeee  3 9746 9762 1123 CAAT 590604 597 613 TTATTGGGCGATC eeekkdddddddkkeee 15 9747 9763 1124 CCAA 590605 598 614 TTTATTGGGCGAT eeekkdddddddkkeee 20 9748 9764 1125 CCCA 590606 599 615 GTTTATTGGGCGA eeekkdddddddkkeee 18 9749 9765 1126 TCCC 590607 600 616 TGTTTATTGGGCG eeekkdddddddkkeee 21 9750 9766 1127 ATCC 590608 601 617 ATGTTTATTGGGC eeekkdddddddkkeee 28 9751 9767 1128 GATC 590609 602 618 AATGTTTATTGGG eeekkdddddddkkeee 30 9752 9768 1129 CGAT 590610 603 619 GAATGTTTATTGG eeekkdddddddkkeee 14 9753 9769 1130 GCGA 590611 604 620 GGAATGTTTATTG eeekkdddddddkkeee 15 9754 9770 1131 GGCG 590612 607 623 AAGGGAATGTTT eeekkdddddddkkeee  2 9757 9773 1132 ATTGG 590613 608 624 CAAGGGAATGTT eeekkdddddddkkeee n.d. 9758 9774 1133 TATTG 590614 609 625 CCAAGGGAATGT eeekkdddddddkkeee n.d. 9759 9775 1134 TTATT 590615 610 626 TCCAAGGGAATG eeekkdddddddkkeee n.d. 9760 9776 1135 TTTAT 590616 611 627 ATCCAAGGGAAT eeekkdddddddkkeee n.d. 9761 9777 1136 GTTTA 590617 612 628 CATCCAAGGGAA eeekkdddddddkkeee n.d. 9762 9778 1137 TGTTT 590618 613 629 ACATCCAAGGGA eeekkdddddddkkeee n.d. 9763 9779 1138 ATGTT 590619 614 630 TACATCCAAGGG eeekkdddddddkkeee n.d. 9764 9780 1139 AATGT 590620 615 631 CTACATCCAAGG eeekkdddddddkkeee n.d. 9765 9781 1140 GAATG 590621 616 632 ACTACATCCAAG eeekkdddddddkkeee  7 9766 9782 1141 GGAAT 590622 617 633 GACTACATCCAA eeekkdddddddkkeee 19 9767 9783 1142 GGGAA 590623 618 634 AGACTACATCCA eeekkdddddddkkeee 39 9768 9784 1143 AGGGA 590624 619 635 CAGACTACATCC eeekkdddddddkkeee 53 9769 9785 1144 AAGGG 590625 620 636 TCAGACTACATCC eeekkdddddddkkeee 57 9770 9786 1145 AAGG 590626 621 637 CTCAGACTACATC eeekkdddddddkkeee 76 9771 9787 1146 CAAG 590627 622 638 CCTCAGACTACAT eeekkdddddddkkeee 58 9772 9788 1147 CCAA 590628 623 639 GCCTCAGACTAC eeekkdddddddkkeee 43 9773 9789 1148 ATCCA 590629 624 640 GGCCTCAGACTA eeekkdddddddkkeee 24 9774 9790 1149 CATCC 590630 625 641 GGGCCTCAGACT eeekkdddddddkkeee 24 9775 9791 1150 ACATC 590631 643 659 GATAACAGATGA eeekkdddddddkkeee 11 9793 9809 1151 GTTAA 590632 644 660 GGATAACAGATG eeekkdddddddkkeee 32 9794 9810 1152 AGTTA 590633 645 661 AGGATAACAGAT eeekkdddddddkkeee 45 9795 9811 1153 GAGTT 590634 646 662 CAGGATAACAGA eeekkdddddddkkeee 65 9796 9812 1154 TGAGT 590635 647 663 GCAGGATAACAG eeekkdddddddkkeee 58 9797 9813 1155 ATGAG 590636 648 664 AGCAGGATAACA eeekkdddddddkkeee 45 9798 9814 1156 GATGA 590637 649 665 TAGCAGGATAAC eeekkdddddddkkeee 34 9799 9815 1157 AGATG 590638 650 666 CTAGCAGGATAA eeekkdddddddkkeee 39 9800 9816 1158 CAGAT 590639 651 667 GCTAGCAGGATA eeekkdddddddkkeee 10 9801 9817 1159 ACAGA 590640 652 668 AGCTAGCAGGAT eeekkdddddddkkeee 15 9802 9818 1160 AACAG 590641 653 669 CAGCTAGCAGGA eeekkdddddddkkeee 21 9803 9819 1161 TAACA 590642 654 670 ACAGCTAGCAGG eeekkdddddddkkeee 20 9804 9820 1162 ATAAC 590643 655 671 TACAGCTAGCAG eeekkdddddddkkeee 40 9805 9821 1163 GATAA 590644 656 672 CTACAGCTAGCA eeekkdddddddkkeee 55 9806 9822 1164 GGATA 590645 657 673 TCTACAGCTAGC eeekkdddddddkkeee 51 9807 9823 1165 AGGAT 590646 658 674 TTCTACAGCTAGC eeekkdddddddkkeee 31 9808 9824 1166 AGGA 590647 659 675 TTTCTACAGCTAG eeekkdddddddkkeee 38 9809 9825 1167 CAGG 590648 660 676 ATTTCTACAGCTA eeekkdddddddkkeee 45 9810 9826 1168 GCAG 590649 661 677 CATTTCTACAGCT eeekkdddddddkkeee 34 9811 9827 1169 AGCA 590650 664 680 ATACATTTCTACA eeekkdddddddkkeee 57 9814 9830 1170 GCTA 590651 665 681 GATACATTTCTAC eeekkdddddddkkeee 40 9815 9831 1171 AGCT 590652 683 699 GTGTTTAATGTTT eeekkdddddddkkeee 37 9833 9849 1172 ATCA 590653 684 700 AGTGTTTAATGTT eeekkdddddddkkeee 67 9834 9850 1173 TATC 590654 685 701 CAGTGTTTAATGT eeekkdddddddkkeee 54 9835 9851 1174 TTAT 590655 686 702 ACAGTGTTTAATG eeekkdddddddkkeee 56 9836 9852 1175 TTTA 590656 687 703 TACAGTGTTTAAT eeekkdddddddkkeee 30 9837 9853 1176 GTTT 590657 688 704 TTACAGTGTTTAA eeekkdddddddkkeee 18 9838 9854 1177 TGTT 590658 689 705 ATTACAGTGTTTA eeekkdddddddkkeee 24 9839 9855 1178 ATGT 590659 690 706 GATTACAGTGTTT eeekkdddddddkkeee 10 9840 9856 1179 AATG 590660 691 707 AGATTACAGTGTT eeekkdddddddkkeee 45 9841 9857 1180 TAAT 590661 692 708 AAGATTACAGTG eeekkdddddddkkeee 34 9842 9858 1181 TTTAA 590662 693 709 TAAGATTACAGT eeekkdddddddkkeee 54 9843 9859 1182 GTTTA 590663 694 710 TTAAGATTACAGT eeekkdddddddkkeee 54 9844 9860 1183 GTTT 590664 772 788 ATCTTCCAAGTGA eeekkdddddddkkeee  7 9922 9938 1184 TCAT 590665 773 789 AATCTTCCAAGTG eeekkdddddddkkeee 23 9923 9939 1185 ATCA 590666 774 790 AAATCTTCCAAGT eeekkdddddddkkeee  4 9924 9940 1186 GATC 590667 775 791 CAAATCTTCCAA eeekkdddddddkkeee 18 9925 9941 1187 GTGAT

TABLE 16 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmers targeting SEQ ID NO: 1 and/or 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence Chemistry inhibition Site Site NO 590512 169 185 CGTCGCCCTT eeekkdddddddkkeee 16   975   991  968 CAGCACG 590668 777 793 TACAAATCTT eeekkdddddddkkeee 17  9927  9943 1188 CCAAGTG 590669 778 794 ATACAAATC eeekkdddddddkkeee 15  9928  9944 1189 TTCCAAGT 590670 779 795 TATACAAAT eeekkdddddddkkeee 11  9929  9945 1190 CTTCCAAG 590671 780 796 CTATACAAA eeekkdddddddkkeee 13  9930  9946 1191 TCTTCCAA 590672 781 797 ACTATACAA eeekkdddddddkkeee 10  9931  9947 1192 ATCTTCCA 590673 782 798 AACTATACA eeekkdddddddkkeee 28  9932  9948 1193 AATCTTCC 590674 783 799 AAACTATAC eeekkdddddddkkeee 26  9933  9949 1194 AAATCTTC 590675 786 802 ATAAAACTA eeekkdddddddkkeee 14  9936  9952 1195 TACAAATC 590676 791 807 GTTTTATAAA eeekkdddddddkkeee 22  9941  9957 1196 ACTATAC 590677 793 809 GAGTTTTATA eeekkdddddddkkeee  6  9943  9959 1197 AAACTAT 590678 814 830 ATTGAAACA eeekkdddddddkkeee 22  9964  9980 1198 GACATTTT 590679 815 831 CATTGAAAC eeekkdddddddkkeee 11  9965  9981 1199 AGACATTT 590680 816 832 TCATTGAAA eeekkdddddddkkeee 10  9966  9982 1200 CAGACATT 590681 817 833 GTCATTGAA eeekkdddddddkkeee 23  9967  9983 1201 ACAGACAT 590682 818 834 GGTCATTGA eeekkdddddddkkeee 11  9968  9984 1202 AACAGACA 590683 819 835 AGGTCATTG eeekkdddddddkkeee 21  9969  9985 1203 AAACAGAC 590684 820 836 CAGGTCATT eeekkdddddddkkeee 14  9970  9986 1204 GAAACAGA 590685 821 837 ACAGGTCAT eeekkdddddddkkeee 14  9971  9987 1205 TGAAACAG 590686 822 838 TACAGGTCA eeekkdddddddkkeee  9  9972  9988 1206 TTGAAACA 590687 823 839 ATACAGGTC eeekkdddddddkkeee 14  9973  9989 1207 ATTGAAAC   590688 824 840 AATACAGGT eeekkdddddddkkeee  6  9974  9990 1208 CATTGAAA 590689 825 841 AAATACAGG eeekkdddddddkkeee  2  9975  9991 1209 TCATTGAA 590690 826 842 AAAATACAG eeekkdddddddkkeee n.d.  9976  9992 1210 GTCATTGA 590691 827 843 CAAAATACA eeekkdddddddkkeee n.d.  9977  9993 1211 GGTCATTG 590692 828 844 GCAAAATAC eeekkdddddddkkeee n.d.  9978  9994 1212 AGGTCATT 590693 829 845 GGCAAAATA eeekkdddddddkkeee n.d.  9979  9995 1213 CAGGTCAT 590694 830 846 TGGCAAAAT eeekkdddddddkkeee n.d.  9980  9996 1214 ACAGGTCA 590695 831 847 CTGGCAAAA eeekkdddddddkkeee n.d.  9981  9997 1215 TACAGGTC 590696 832 848 TCTGGCAAA eeekkdddddddkkeee n.d.  9982  9998 1216 ATACAGGT 590697 833 849 GTCTGGCAA eeekkdddddddkkeee n.d.  9983  9999 1217 AATACAGG 590698 834 850 AGTCTGGCA eeekkdddddddkkeee  1  9984 10000 1218 AAATACAG 590699 835 851 AAGTCTGGC eeekkdddddddkkeee 10  9985 10001 1219 AAAATACA 590700 836 852 TAAGTCTGG eeekkdddddddkkeee  4  9986 10002 1220 CAAAATAC 590701 837 853 TTAAGTCTGG eeekkdddddddkkeee  2  9987 10003 1221 CAAAATA 590702 853 869 AATACCCAT eeekkdddddddkkeee  7 10003 10019 1222 CTGTGATT 590703 854 870 TAATACCCAT eeekkdddddddkkeee  4 10004 10020 1223 CTGTGAT 590704 855 871 TTAATACCCA eeekkdddddddkkeee  2 10005 10021 1224 TCTGTGA 590705 856 872 TTTAATACCC eeekkdddddddkkeee  0 10006 10022 1225 ATCTGTG 590706 857 873 GTTTAATACC eeekkdddddddkkeee 17 10007 10023 1226 CATCTGT 590707 858 874 AGTTTAATAC eeekkdddddddkkeee 10 10008 10024 1227 CCATCTG 590708 859 875 AAGTTTAAT eeekkdddddddkkeee 12 10009 10025 1228 ACCCATCT 590709 860 876 CAAGTTTAAT eeekkdddddddkkeee 37 10010 10026 1229 ACCCATC 590710 861 877 ACAAGTTTA eeekkdddddddkkeee 23 10011 10027 1230 ATACCCAT 590711 862 878 GACAAGTTT eeekkdddddddkkeee 24 10012 10028 1231 AATACCCA 590712 863 879 TGACAAGTTT eeekkdddddddkkeee 27 10013 10029 1232 AATACCC 590713 864 880 CTGACAAGT eeekkdddddddkkeee 10 10014 10030 1233 TTAATACC 590714 865 881 TCTGACAAG eeekkdddddddkkeee  0 10015 10031 1234 TTTAATAC 590715 866 882 TTCTGACAA eeekkdddddddkkeee  6 10016 10032 1235 GTTTAATA 590716 867 883 ATTCTGACA eeekkdddddddkkeee  9 10017 10033 1236 AGTTTAAT 590717 868 884 AATTCTGAC eeekkdddddddkkeee 15 10018 10034 1237 AAGTTTAA 590718 869 885 AAATTCTGA eeekkdddddddkkeee 21 10019 10035 1238 CAAGTTTA 590719 870 886 GAAATTCTG eeekkdddddddkkeee 14 10020 10036 1239 ACAAGTTT 590720 871 887 AGAAATTCT eeekkdddddddkkeee  8 10021 10037 1240 GACAAGTT 590721 872 888 AAGAAATTC eeekkdddddddkkeee 18 10022 10038 1241 TGACAAGT 590722 891 907 TTCACAGGCT eeekkdddddddkkeee  9 10041 10057 1242 TGAATGA 590723 892 908 ATTCACAGG eeekkdddddddkkeee 11 10042 10058 1243 CTTGAATG 590724 893 909 TATTCACAG eeekkdddddddkkeee  0 10043 10059 1244 GCTTGAAT 590725 894 910 TTATTCACAG eeekkdddddddkkeee 10 10044 10060 1245 GCTTGAA 590726 895 911 TTTATTCACA eeekkdddddddkkeee 29 10045 10061 1246 GGCTTGA 590727 896 912 TTTTATTCAC eeekkdddddddkkeee 28 10046 10062 1247 AGGCTTG 590728 897 913 TTTTTATTCA eeekkdddddddkkeee 31 10047 10063 1248 CAGGCTT 590729 898 914 GTTTTTATTC eeekkdddddddkkeee 10 10048 10064 1249 ACAGGCT 590731 899 915 GGTTTTTATT eeekkdddddddkkeee 22 10049 10065 1250 CACAGGC 590732 900 916 GGGTTTTTAT eeekkdddddddkkeee 17 10050 10066 1251 TCACAGG 590733 901 917 AGGGTTTTTA eeekkdddddddkkeee 24 10051 10067 1252 TTCACAG 590734 902 918 CAGGGTTTTT eeekkdddddddkkeee 17 10052 10068 1253 ATTCACA 590735 903 919 ACAGGGTTTT eeekkdddddddkkeee 10 10053 10069 1254 TATTCAC 590736 904 920 TACAGGGTTT eeekkdddddddkkeee 11 10054 10070 1255 TTATTCA 590737 905 921 ATACAGGGT eeekkdddddddkkeee  3 10055 10071 1256 TTTTATTC 590738 906 922 CATACAGGG eeekkdddddddkkeee  0 10056 10072 1257 TTTTTATT 590739 907 923 CCATACAGG eeekkdddddddkkeee  1 10057 10073 1258 GTTTTTAT 590740 908 924 GCCATACAG eeekkdddddddkkeee 11 10058 10074 1259 GGTTTTTA 590741 909 925 TGCCATACA eeekkdddddddkkeee  9 10059 10075 1260 GGGTTTTT 590742 910 926 GTGCCATAC eeekkdddddddkkeee  7 10060 10076 1261 AGGGTTTT 590743 911 927 AGTGCCATA eeekkdddddddkkeee  9 10061 10077 1262 CAGGGTTT 590744 938 954 TTGGATTCTT eeekkdddddddkkeee  8 10088 10104 1263 TTAATAG 590745 951 967 TTTTAGTTTG eeekkdddddddkkeee 12 n/a n/a 1264 AATTTGG

TABLE 17 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmers targeting SEQ ID NO: 1 and/or 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence Chemistry inhibition Site Site NO 333611 167 186 CCGTCGCCCT eeeeeddddddddddeeeee 66  973  992   21 TCAGCACGCA 590067 202 221 CCTTCTGCTCG eeeeeddddddddddeeeee 53 1008 1027  120 AAATTGATG 590074 209 228 TTACTTTCCTT eeeeeddddddddddeeeee 30 n/a n/a  127 CTGCTCGAA 590457 211 227 TACTTTCCTTC eeekkdddddddkkeee 14 n/a n/a  980 TGCTCG 590458 212 228 TTACTTTCCTT eeekkdddddddkkeee 22 n/a n/a  981 CTGCTC 590459 213 229 ATTACTTTCCT eeekkdddddddkkeee 15 n/a n/a  982 TCTGCT 590461 214 230 CATTACTTTCC eeekkdddddddkkeee 28 n/a n/a  983 TTCTGC 590462 215 231 CCATTACTTTC eeekkdddddddkkeee 37 n/a n/a  984 CTTCTG 590463 216 232 TCCATTACTTT eeekkdddddddkkeee 18 n/a n/a  985 CCTTCT 590082 217 236 CTGGTCCATT eeeeeddddddddddeeeee 50 n/a n/a  135 ACTTTCCTTC 590464 217 233 GTCCATTACTT eeekkdddddddkkeee 33 n/a n/a  986 TCCTTC 590465 218 234 GGTCCATTAC eeekkdddddddkkeee 18 4972 4988  987 TTTCCTT 590130 363 382 TTCATCCTTTG eeeeeddddddddddeeeee 30 7679 7698  194 GCCCACCGT 590536 363 379 ATCCTTTGGC eeekkdddddddkkeee 51 7679 7695 1056 CCACCGT 590537 364 380 CATCCTTTGG eeekkdddddddkkeee 38 7680 7696 1057 CCCACCG 590538 365 381 TCATCCTTTGG eeekkdddddddkkeee 27 7681 7697 1058 CCCACC 590539 366 382 TTCATCCTTTG eeekkdddddddkkeee 26 7682 7698 1059 GCCCAC 590540 367 383 CTTCATCCTTT eeekkdddddddkkeee 35 7683 7699 1060 GGCCCA 590541 368 384 TCTTCATCCTT eeekkdddddddkkeee 15 7684 7700 1061 TGGCCC 590542 369 385 CTCTTCATCCT eeekkdddddddkkeee 26 7685 7701 1062 TTGGCC 590543 370 386 TCTCTTCATCC eeekkdddddddkkeee 14 7686 7702 1063 TTTGGC 590138 371 390 TGCCTCTCTTC eeeeeddddddddddeeeee 32 n/a n/a  202 ATCCTTTGG 590146 505 524 CATCTGCTTTT eeeeeddddddddddeeeee 46 9655 9674  211 TCATGGACC 590613 608 624 CAAGGGAATG eeekkdddddddkkeee 19 9758 9774 1133 TTTATTG 590614 609 625 CCAAGGGAAT eeekkdddddddkkeee 36 9759 9775 1134 GTTTATT 590615 610 626 TCCAAGGGAA eeekkdddddddkkeee 32 9760 9776 1135 TGTTTAT 590616 611 627 ATCCAAGGGA eeekkdddddddkkeee 42 9761 9777 1136 ATGTTTA 590617 612 628 CATCCAAGGG eeekkdddddddkkeee 16 9762 9778 1137 AATGTTT 590618 613 629 ACATCCAAGG eeekkdddddddkkeee 30 9763 9779 1138 GAATGTT 590619 614 630 TACATCCAAG eeekkdddddddkkeee 30 9764 9780 1139 GGAATGT 590620 615 631 CTACATCCAA eeekkdddddddkkeee 40 9765 9781 1140 GGGAATG 590690 826 842 AAAATACAGG eeekkdddddddkkeee 28 9976 9992 1210 TCATTGA 590691 827 843 CAAAATACAG eeekkdddddddkkeee 23 9977 9993 1211 GTCATTG 590692 828 844 GCAAAATACA eeekkdddddddkkeee 43 9978 9994 1212 GGTCATT 590693 829 845 GGCAAAATAC eeekkdddddddkkeee 39 9979 9995 1213 AGGTCAT 590694 830 846 TGGCAAAATA eeekkdddddddkkeee 26 9980 9996 1214 CAGGTCA 590695 831 847 CTGGCAAAAT eeekkdddddddkkeee 18 9981 9997 1215 ACAGGTC 590696 832 848 TCTGGCAAAA eeekkdddddddkkeee 0 9982 9998 1216 TACAGGT 590697 833 849 GTCTGGCAAA eeekkdddddddkkeee 24 9983 9999 1217 ATACAGG

Example 4: Inhibition of Human SOD-1 in HepG2 Cells by Deoxy, MOE and cEt Gapmers

Modified oligonucleotides were designed targeting a superoxide dismutase 1, soluble (SOD-1) nucleic acid and were tested for their effects on SOD-1 mRNA in vitro. ISIS 333611, a5-10-5 MOE gapmer which was previously described in WO2005/040180, was included as a benchmark.

The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 4,000 nM modified oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR.

Human primer probe set RTS3898 was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells. ‘n.d.’ indicates that inhibition levels were not measured.

The newly designed modified oligonucleotides in the Tables below were designed as deoxy, MOE, and cEt gapmers or 5-10-5 gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment is comprised of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a2′-MOE modification. The deoxy, MOE and cEt oligonucleotides are 17 nucleosides in length wherein the nucleoside has a MOE sugar modification, a cEt sugar modification, or a deoxy moiety. The sugar chemistry of each oligonucleotide is denoted as in the Chemistry column, where ‘k’ indicates a cEt modified sugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’ indicates a 2′-MOE modified sugar. The internucleoside linkages throughout each gapmer are phosphorothioate linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. Each gapmer listed in the Tables below is targeted to either the human SOD-1 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the human SOD-1 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000 to 18704000). ‘n/a’ indicates that the modified oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 18 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and/or 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence inhibition Site Site NO 596168   3  22 CTCGCCCACTCTGGCCCCAA 45  809  828 1265 596169   5  24 GCCTCGCCCACTCTGGCCCC 37  811  830 1266 596170   7  26 GCGCCTCGCCCACTCTGGCC 33  813  832 1267 596171   9  28 CCGCGCCTCGCCCACTCTGG 27  815  834 1268 596172  11  30 CTCCGCGCCTCGCCCACTCT 40  817  836 1269 596173  13  32 ACCTCCGCGCCTCGCCCACT 77  819  838 1270 596174  15  34 AGACCTCCGCGCCTCGCCCA 72  821  840 1271 596175  17  36 CCAGACCTCCGCGCCTCGCC 46  823  842 1272 596176  19  38 GGCCAGACCTCCGCGCCTCG 49  825  844 1273 150508  21  40 TAGGCCAGACCTCCGCGCCT 33  827  846  107 596177  23  42 TATAGGCCAGACCTCCGCGC 40  829  848 1274 596178  25  44 TTTATAGGCCAGACCTCCGC 69  831  850 1275 150509  27  46 ACTTTATAGGCCAGACCTCC 64  833  852  108 596179  31  50 GACTACTTTATAGGCCAGAC 74  837  856 1276 596180  33  52 GCGACTACTTTATAGGCCAG 19  839  858 1277 596181  37  56 CTCCGCGACTACTTTATAGG 27  843  862 1278 596182  39  58 GTCTCCGCGACTACTTTATA 22  845  864 1279 596183  41  60 CCGTCTCCGCGACTACTTTA 20  847  866 1280 596184  43  62 CCCCGTCTCCGCGACTACTT 16  849  868 1281 596185  45  64 CACCCCGTCTCCGCGACTAC 13  851  870 1282 596186  47  66 AGCACCCCGTCTCCGCGACT 24  853  872 1283 596187  49  68 CCAGCACCCCGTCTCCGCGA 38  855  874 1284 596188  51  70 AACCAGCACCCCGTCTCCGC 11  857  876 1285 596189  53  72 CAAACCAGCACCCCGTCTCC 13  859  878 1286 596190  55  74 CGCAAACCAGCACCCCGTCT 21  861  880 1287 150510  57  76 GACGCAAACCAGCACCCCGT 45  863  882  109 596191  59  78 ACGACGCAAACCAGCACCCC 30  865  884 1288 596192  61  80 CTACGACGCAAACCAGCACC 19  867  886 1289 596193  63  82 GACTACGACGCAAACCAGCA 40  869  888 1290 596194  65  84 GAGACTACGACGCAAACCAG 23  871  890 1291 596195  67  86 AGGAGACTACGACGCAAACC 35  873  892 1292 596196  69  88 GCAGGAGACTACGACGCAAA 33  875  894 1293 596197  71  90 CTGCAGGAGACTACGACGCA 36  877  896 1294 596198  73  92 CGCTGCAGGAGACTACGACG 23  879  898 1295 596199  91 110 TGCAACGGAAACCCCAGACG 21  897  916 1296 596200  93 112 ACTGCAACGGAAACCCCAGA 43  899  918 1297 596201  97 116 GAGGACTGCAACGGAAACCC 24  903  922 1298 596202  99 118 CCGAGGACTGCAACGGAAAC 29  905  924 1299 596203 101 120 TTCCGAGGACTGCAACGGAA  5  907  926 1300 150438 103 122 GGTTCCGAGGACTGCAACGG 35  909  928  110 345716 105 124 CTGGTTCCGAGGACTGCAAC 51  911  930 1301 150439 107 126 TCCTGGTTCCGAGGACTGCA 24  913  932  111 596204 109 128 GGTCCTGGTTCCGAGGACTG 14  915  934 1302 150440 111 130 GAGGTCCTGGTTCCGAGGAC 31  917  936  112 596205 113 132 CCGAGGTCCTGGTTCCGAGG 18  919  938 1303 345718 115 134 CGCCGAGGTCCTGGTTCCGA 24  921  940 1304 596206 117 136 CACGCCGAGGTCCTGGTTCC 23  923  942 1305 596207 119 138 GCCACGCCGAGGTCCTGGTT 38  925  944 1306 596208 123 142 CTAGGCCACGCCGAGGTCCT 39  929  948 1307 345720 125 144 CGCTAGGCCACGCCGAGGTC 52  931  950 1308 596209 127 146 CTCGCTAGGCCACGCCGAGG 46  933  952 1309 596210 129 148 AACTCGCTAGGCCACGCCGA 44  935  954 1310 596211 131 150 ATAACTCGCTAGGCCACGCC 12  937  956 1311 596212 133 152 CCATAACTCGCTAGGCCACG 22  939  958 1312 345722 135 154 CGCCATAACTCGCTAGGCCA 59  941  960 1313 150442 137 156 GTCGCCATAACTCGCTAGGC 40  943  962  113 146143 157 176 TCAGCACGCACACGGCCTTC 52  963  982  114 195753 159 178 CTTCAGCACGCACACGGCCT 57  965  984  115 333607 161 180 CCCTTCAGCACGCACACGGC 37  967  986  116 333608 163 182 CGCCCTTCAGCACGCACACG 23  969  988  117 333611 167 186 CCGTCGCCCTTCAGCACGCA 67  973  992   21 596213 171 190 TGGGCCGTCGCCCTTCAGCA 12  977  996 1314 596214 173 192 ACTGGGCCGTCGCCCTTCAG 26  979  998 1315 596215 175 194 GCACTGGGCCGTCGCCCTTC 14  981 1000 1316 596216 177 196 CTGCACTGGGCCGTCGCCCT 24  983 1002 1317 596217 181 200 TGCCCTGCACTGGGCCGTCG 38  987 1006 1318 596218 183 202 GATGCCCTGCACTGGGCCGT 15  989 1008 1319 596219 185 204 ATGATGCCCTGCACTGGGCC 20  991 1010 1320 596220 189 208 ATTGATGATGCCCTGCACTG  8  995 1014 1321 596221 191 210 AAATTGATGATGCCCTGCAC 14  997 1016 1322 596222 193 212 CGAAATTGATGATGCCCTGC 32  999 1018 1323 596223 195 214 CTCGAAATTGATGATGCC CT 31 1001 1020 1324 596224 197 216 TGCTCGAAATTGATGATGCC 20 1003 1022 1325 596225 199 218 TCTGCTCGAAATTGATGATG 14 1005 1024 1326 596226 201 220 CTTCTGCTCGAAATTGATGA 11 1007 1026 1327 596227 240 259 TTTAATGCTTCCCCACACCT 15 4994 5013 1328 596228 242 261 CCTTTAATGCTTCCCCACAC  1 4996 5015 1329 596229 244 263 GTCCTTTAATGCTTCCCCAC  9 4998 5017 1330

TABLE 19 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmers targeting SEQ ID NO: 1 and/or 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence Chemistry inhibition Site Site NO 596530 164 180 CCCTTCAGCA eekkddddddddkkeee 74  970  986 1331 CGCACAC 596721 164 180 CCCTTCAGCA eekkdddddddddkkee 81  970  986 1331 CGCACAC 596531 165 181 GCCCTTCAGC eekkddddddddkkeee 75  971  987 1332 ACGCACA 596722 165 181 GCCCTTCAGC eekkdddddddddkkee 60  971  987 1332 ACGCACA 596532 166 182 CGCCCTTCAG eekkddddddddkkeee 67  972  988 1333 CACGCAC 596723 166 182 CGCCCTTCAG eekkdddddddddkkee 73  972  988 1333 CACGCAC 333611 167 186 CCGTCGCCCT eeeeeddddddddddeeeee 73  973  992   21 TCAGCACGCA 596720 167 183 TCGCCCTTCA eekkddddddddkkeee 56  973  989  966 GCACGCA 596911 167 183 TCGCCCTTCA eekkdddddddddkkee 63  973  989  966 GCACGCA 596533 168 184 GTCGCCCTTC eekkddddddddkkeee 60  974  990  967 AGCACGC 596724 168 184 GTCGCCCTTC eekkdddddddddkkee 72  974  990  967 AGCACGC 596534 169 185 CGTCGCCCTT eekkddddddddkkeee 52  975  991  968 CAGCACG 596725 169 185 CGTCGCCCTT eekkdddddddddkkee 43  975  991  968 CAGCACG 596535 170 186 CCGTCGCCCT eekkddddddddkkeee 71  976  992  969 TCAGCAC 596726 170 186 CCGTCGCCCT eekkdddddddddkkee 75  976  992  969 TCAGCAC 596536 171 187 GCCGTCGCCC eekkddddddddkkeee 64  977  993  970 TTCAGCA 596727 171 187 GCCGTCGCCC eekkdddddddddkkee 57  977  993  970 TTCAGCA 596537 577 593 CACCACAAGC eekkddddddddkkeee 48 9727 9743 1334 CAAACGA 596728 577 593 CACCACAAGC eekkdddddddddkkee 46 9727 9743 1334 CAAACGA 596538 578 594 ACACCACAAG eekkddddddddkkeee 27 9728 9744 1335 CCAAACG 596729 578 594 ACACCACAAG eekkdddddddddkkee 45 9728 9744 1335 CCAAACG 596539 579 595 TACACCACAA eekkddddddddkkeee 56 9729 9745 1336 GCCAAAC 596730 579 595 TACACCACAA eekkdddddddddkkee 63 9729 9745 1336 GCCAAAC 596540 580 596 TTACACCACA eekkddddddddkkeee 60 9730 9746 1337 AGCCAAA 596731 580 596 TTACACCACA eekkdddddddddkkee 63 9730 9746 1337 AGCCAAA 596541 581 597 ATTACACCAC eekkddddddddkkeee 46 9731 9747 1338 AAGCCAA 596732 581 597 ATTACACCAC eekkdddddddddkkee 63 9731 9747 1338 AAGCCAA 596542 582 598 AATTACACCA eekkddddddddkkeee 62 9732 9748 1111 CAAGCCA 596733 582 598 AATTACACCA eekkdddddddddkkee 56 9732 9748 1111 CAAGCCA 596543 583 599 CAATTACACC eekkddddddddkkeee 58 9733 9749 1112 ACAAGCC 596734 583 599 CAATTACACC eekkdddddddddkkee 61 9733 9749 1112 ACAAGCC 596544 584 600 CCAATTACAC eekkddddddddkkeee 66 9734 9750 1113 CACAAGC 596735 584 600 CCAATTACAC eekkdddddddddkkee 73 9734 9750 1113 CACAAGC 596545 585 601 CCCAATTACA eekkddddddddkkeee 63 9735 9751 1114 CCACAAG 596736 585 601 CCCAATTACA eekkdddddddddkkee 74 9735 9751 1114 CCACAAG 596546 588 604 GATCCCAATT eekkddddddddkkeee 41 9738 9754 1115 ACACCAC 596737 588 604 GATCCCAATT eekkdddddddddkkee 58 9738 9754 1115 ACACCAC 596547 589 605 CGATCCCAAT eekkddddddddkkeee 57 9739 9755 1116 TACACCA 596738 589 605 CGATCCCAAT eekkdddddddddkkee 59 9739 9755 1116 TACACCA 596548 590 606 GCGATCCCAA eekkddddddddkkeee 31 9740 9756 1117 TTACACC 596739 590 606 GCGATCCCAA eekkdddddddddkkee 58 9740 9756 1117 TTACACC 596549 591 607 GGCGATCCCA eekkddddddddkkeee 33 9741 9757 1118 ATTACAC 596740 591 607 GGCGATCCCA eekkdddddddddkkee 66 9741 9757 1118 ATTACAC 596550 592 608 GGGCGATCCC eekkddddddddkkeee 30 9742 9758 1119 AATTACA 596741 592 608 GGGCGATCCC eekkdddddddddkkee 30 9742 9758 1119 AATTACA 596551 593 609 TGGGCGATCC eekkddddddddkkeee 19 9743 9759 1120 CAATTAC 596742 593 609 TGGGCGATCC eekkdddddddddkkee 46 9743 9759 1120 CAATTAC 596552 594 610 TTGGGCGATC eekkddddddddkkeee 14 9744 9760 1121 CCAATTA 596743 594 610 TTGGGCGATC eekkdddddddddkkee  5 9744 9760 1121 CCAATTA 596553 595 611 ATTGGGCGAT eekkddddddddkkeee  2 9745 9761 1122 CCCAATT 596744 595 611 ATTGGGCGAT eekkdddddddddkkee 23 9745 9761 1122 CCCAATT 596554 596 612 TATTGGGCGA eekkddddddddkkeee 19 9746 9762 1123 TCCCAAT 596745 596 612 TATTGGGCGA eekkdddddddddkkee  6 9746 9762 1123 TCCCAAT 596555 597 613 TTATTGGGCG eekkddddddddkkeee 41 9747 9763 1124 ATCCCAA 596746 597 613 TTATTGGGCG eekkdddddddddkkee 41 9747 9763 1124 ATCCCAA 596556 598 614 TTTATTGGGC eekkddddddddkkeee 34 9748 9764 1125 GATCCCA 596747 598 614 TTTATTGGGC eekkdddddddddkkee 46 9748 9764 1125 GATCCCA 596557 599 615 GTTTATTGGG eekkddddddddkkeee 54 9749 9765 1126 CGATCCC 596748 599 615 GTTTATTGGG eekkdddddddddkkee 68 9749 9765 1126 CGATCCC 596558 600 616 TGTTTATTGG eekkddddddddkkeee 50 9750 9766 1127 GCGATCC 596749 600 616 TGTTTATTGG eekkdddddddddkkee 47 9750 9766 1127 GCGATCC 596559 601 617 ATGTTTATTG eekkddddddddkkeee 76 9751 9767 1128 GGCGATC 596750 601 617 ATGTTTATTG eekkdddddddddkkee 64 9751 9767 1128 GGCGATC 596560 602 618 AATGTTTATT eekkddddddddkkeee 61 9752 9768 1129 GGGCGAT 596751 602 618 AATGTTTATT eekkdddddddddkkee 64 9752 9768 1129 GGGCGAT 596561 603 619 GAATGTTTAT eekkddddddddkkeee 47 9753 9769 1130 TGGGCGA 596752 603 619 GAATGTTTAT eekkdddddddddkkee 65 9753 9769 1130 TGGGCGA 596562 604 620 GGAATGTTTA eekkddddddddkkeee 37 9754 9770 1131 TTGGGCG 596753 604 620 GGAATGTTTA eekkdddddddddkkee 58 9754 9770 1131 TTGGGCG 596563 608 624 CAAGGGAATG eekkddddddddkkeee 43 9758 9774 1133 TTTATTG 596754 608 624 CAAGGGAATG eekkdddddddddkkee 38 9758 9774 1133 TTTATTG 596564 609 625 CCAAGGGAAT eekkddddddddkkeee 57 9759 9775 1134 GTTTATT 596755 609 625 CCAAGGGAAT eekkdddddddddkkee 52 9759 9775 1134 GTTTATT 596565 610 626 TCCAAGGGAA eekkddddddddkkeee 27 9760 9776 1135 TGTTTAT 596756 610 626 TCCAAGGGAA eekkdddddddddkkee 57 9760 9776 1135 TGTTTAT 596566 611 627 ATCCAAGGGA eekkddddddddkkeee 35 9761 9777 1136 ATGTTTA 596757 611 627 ATCCAAGGGA eekkdddddddddkkee 39 9761 9777 1136 ATGTTTA 596567 616 632 ACTACATCCA eekkddddddddkkeee 42 9766 9782 1141 AGGGAAT 596758 616 632 ACTACATCCA eekkdddddddddkkee 48 9766 9782 1141 AGGGAAT

TABLE 20 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmers targeting SEQ ID NO: 1 and/or 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence Chemistry inhibition Site Site NO 333611 167 186 CCGTCGCCCT eeeeeddddddddddeeeee 64  973  992   21 TCAGCACGCA 596720 167 183 TCGCCCTTCA eekkddddddddkkeee 56  973  989  966 GCACGCA 596911 167 183 TCGCCCTTCA eekkdddddddddkkee 60  973  989  966 GCACGCA 596568 617 633 GACTACATCC eekkddddddddkkeee 50 9767 9783 1142 AAGGGAA 596759 617 633 GACTACATCC eekkdddddddddkkee 57 9767 9783 1142 AAGGGAA 596569 618 634 AGACTACATC eekkddddddddkkeee 53 9768 9784 1143 CAAGGGA 596760 618 634 AGACTACATC eekkdddddddddkkee 55 9768 9784 1143 CAAGGGA 596570 619 635 CAGACTACAT eekkddddddddkkeee 81 9769 9785 1144 CCAAGGG 596761 619 635 CAGACTACAT eekkdddddddddkkee 78 9769 9785 1144 CCAAGGG 596571 620 636 TCAGACTACA eekkddddddddkkeee 79 9770 9786 1145 TCCAAGG 596762 620 636 TCAGACTACA eekkdddddddddkkee 78 9770 9786 1145 TCCAAGG 596572 621 637 CTCAGACTAC eekkddddddddkkeee 85 9771 9787 1146 ATCCAAG 596763 621 637 CTCAGACTAC eekkdddddddddkkee 76 9771 9787 1146 ATCCAAG 596573 622 638 CCTCAGACTA eekkddddddddkkeee 73 9772 9788 1147 CATCCAA 596764 622 638 CCTCAGACTA eekkdddddddddkkee 87 9772 9788 1147 CATCCAA 596574 623 639 GCCTCAGACT eekkddddddddkkeee 69 9773 9789 1148 ACATCCA 596765 623 639 GCCTCAGACT eekkdddddddddkkee 82 9773 9789 1148 ACATCCA 596575 624 640 GGCCTCAGAC eekkddddddddkkeee 70 9774 9790 1149 TACATCC 596766 624 640 GGCCTCAGAC eekkdddddddddkkee 76 9774 9790 1149 TACATCC 596576 625 641 GGGCCTCAGA eekkddddddddkkeee 55 9775 9791 1150 CTACATC 596767 625 641 GGGCCTCAGA eekkdddddddddkkee 58 9775 9791 1150 CTACATC 596577 640 656 AACAGATGA eekkddddddddkkeee 73 9790 9806 1339 GTTAAGGG 596768 640 656 AACAGATGA eekkdddddddddkkee 86 9790 9806 1339 GTTAAGGG 596578 641 657 TAACAGATGA eekkddddddddkkeee 68 9791 9807 1340 GTTAAGG 596769 641 657 TAACAGATGA eekkdddddddddkkee 80 9791 9807 1340 GTTAAGG 596579 642 658 ATAACAGATG eekkddddddddkkeee 27 9792 9808 1341 AGTTAAG 596770 642 658 ATAACAGATG eekkdddddddddkkee 42 9792 9808 1341 AGTTAAG 596580 643 659 GATAACAGAT eekkddddddddkkeee 41 9793 9809 1151 GAGTTAA 596771 643 659 GATAACAGAT eekkdddddddddkkee 28 9793 9809 1151 GAGTTAA 596581 644 660 GGATAACAG eekkddddddddkkeee 63 9794 9810 1152 ATGAGTTA 596772 644 660 GGATAACAG eekkdddddddddkkee 63 9794 9810 1152 ATGAGTTA 596582 645 661 AGGATAACA eekkddddddddkkeee 84 9795 9811 1153 GATGAGTT 596773 645 661 AGGATAACA eekkdddddddddkkee 86 9795 9811 1153 GATGAGTT 596583 646 662 CAGGATAAC eekkddddddddkkeee 95 9796 9812 1154 AGATGAGT 596774 646 662 CAGGATAAC eekkdddddddddkkee 96 9796 9812 1154 AGATGAGT 596584 647 663 GCAGGATAA eekkddddddddkkeee 79 9797 9813 1155 CAGATGAG 596775 647 663 GCAGGATAA eekkdddddddddkkee 86 9797 9813 1155 CAGATGAG 596585 651 667 GCTAGCAGG eekkddddddddkkeee 19 9801 9817 1159 ATAACAGA 596776 651 667 GCTAGCAGG eekkdddddddddkkee 43 9801 9817 1159 ATAACAGA 596586 652 668 AGCTAGCAG eekkddddddddkkeee 57 9802 9818 1160 GATAACAG 596777 652 668 AGCTAGCAG eekkdddddddddkkee 54 9802 9818 1160 GATAACAG 596587 653 669 CAGCTAGCAG eekkddddddddkkeee 71 9803 9819 1161 GATAACA 596778 653 669 CAGCTAGCAG eekkdddddddddkkee 61 9803 9819 1161 GATAACA 596588 654 670 ACAGCTAGCA eekkddddddddkkeee 79 9804 9820 1162 GGATAAC 596779 654 670 ACAGCTAGCA eekkdddddddddkkee 83 9804 9820 1162 GGATAAC 596589 655 671 TACAGCTAGC eekkddddddddkkeee 85 9805 9821 1163 AGGATAA 596780 655 671 TACAGCTAGC eekkdddddddddkkee 86 9805 9821 1163 AGGATAA 596590 656 672 CTACAGCTAG eekkddddddddkkeee 87 9806 9822 1164 CAGGATA 596781 656 672 CTACAGCTAG eekkdddddddddkkee 91 9806 9822 1164 CAGGATA 596591 657 673 TCTACAGCTA eekkddddddddkkeee 75 9807 9823 1165 GCAGGAT 596782 657 673 TCTACAGCTA eekkdddddddddkkee 83 9807 9823 1165 GCAGGAT 596592 658 674 TTCTACAGCT eekkddddddddkkeee 74 9808 9824 1166 AGCAGGA 596783 658 674 TTCTACAGCT eekkdddddddddkkee 79 9808 9824 1166 AGCAGGA 596593 659 675 TTTCTACAGC eekkddddddddkkeee 76 9809 9825 1167 TAGCAGG 596784 659 675 TTTCTACAGC eekkdddddddddkkee 84 9809 9825 1167 TAGCAGG 596594 660 676 ATTTCTACAG eekkddddddddkkeee 66 9810 9826 1168 CTAGCAG 596785 660 676 ATTTCTACAG eekkdddddddddkkee 75 9810 9826 1168 CTAGCAG 596595 665 681 GATACATTTC eekkddddddddkkeee 64 9815 9831 1171 TACAGCT 596786 665 681 GATACATTTC eekkdddddddddkkee 77 9815 9831 1171 TACAGCT 596596 666 682 GGATACATTT eekkddddddddkkeee 75 9816 9832 1342 CTACAGC 596787 666 682 GGATACATTT eekkdddddddddkkee 84 9816 9832 1342 CTACAGC 596597 667 683 AGGATACATT eekkddddddddkkeee 60 9817 9833 1343 TCTACAG 596788 667 683 AGGATACATT eekkdddddddddkkee 77 9817 9833 1343 TCTACAG 596598 668 684 CAGGATACAT eekkddddddddkkeee 79 9818 9834 1344 TTCTACA 596789 668 684 CAGGATACAT eekkdddddddddkkee 85 9818 9834 1344 TTCTACA 596599 672 688 TTATCAGGAT eekkddddddddkkeee 57 9822 9838 1345 ACATTTC 596790 672 688 TTATCAGGAT eekkdddddddddkkee 67 9822 9838 1345 ACATTTC 596600 674 690 GTTTATCAGG eekkddddddddkkeee 85 9824 9840 1346 ATACATT 596791 674 690 GTTTATCAGG eekkdddddddddkkee 88 9824 9840 1346 ATACATT 596601 675 691 TGTTTATCAG eekkddddddddkkeee 70 9825 9841 1347 GATACAT 596792 675 691 TGTTTATCAG eekkdddddddddkkee 83 9825 9841 1347 GATACAT 596602 676 692 ATGTTTATCA eekkddddddddkkeee 85 9826 9842 1348 GGATACA 596793 676 692 ATGTTTATCA eekkdddddddddkkee 81 9826 9842 1348 GGATACA 596603 677 693 AATGTTTATC eekkddddddddkkeee 89 9827 9843 1349 AGGATAC 596794 677 693 AATGTTTATC eekkdddddddddkkee 90 9827 9843 1349 AGGATAC 596604 678 694 TAATGTTTAT eekkddddddddkkeee 90 9828 9844 1350 CAGGATA 596795 678 694 TAATGTTTAT eekkdddddddddkkee 85 9828 9844 1350 CAGGATA 596605 679 695 TTAATGTTTA eekkddddddddkkeee 90 9829 9845 1351 TCAGGAT 596796 679 695 TTAATGTTTA eekkdddddddddkkee 92 9829 9845 1351 TCAGGAT

Example 5: Inhibition of Human SOD-1 in HepG2 Cells by Deoxy, MOE and cEt Gapmers

Modified oligonucleotides were designed targeting an SOD-1 nucleic acid and were tested for their effects on SOD-1 mRNA in vitro. ISIS 333611, a5-10-5 MOE gapmer, which was previously described in WO 2005/040180, was included as a benchmark.

The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 5,000 nM modified oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR.

Human primer probe set RTS3898 was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells. ‘n.d.’ indicates that inhibition levels were not measured.

The newly designed modified oligonucleotides in the Tables below were designed as deoxy, MOE, and cEt gapmers. The gapmers are 17 nucleosides in length wherein each nucleoside has a MOE sugar modification, cEt sugar modification, or a deoxy moiety. The sugar chemistry of each oligonucleotide is denoted as in the Chemistry column, where ‘k’ indicates a cEt modified sugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’ indicates a 2′-MOE modified sugar. The internucleoside linkages throughout each gapmer are phosphorothioate linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. Each gapmer listed in the Tables below is targeted to either the human SOD-1 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the human SOD-1 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000 to 18704000). ‘n/a’ indicates that the modified oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 21  Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO. 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence Chemistry inhibition Site Site NO 333611 167 186 CCGTCGCCCTT eeeeeddddddddddeeeee 71  973  992   21 CAGCACGCA 596720 167 183 TCGCCCTTCAG eekkddddddddkkeee 53  973  989  966 CACGCA 596911 167 183 TCGCCCTTCAG eekkdddddddddkkee 61  973  989  966 CACGCA 596606 681 697 GTTTAATGTTT eekkddddddddkkeee 87 9831 9847 1352 ATCAGG 596797 681 697 GTTTAATGTTT eekkdddddddddkkee 92 9831 9847 1352 ATCAGG 596607 683 699 GTGTTTAATGT eekkddddddddkkeee 86 9833 9849 1172 TTATCA 596798 683 699 GTGTTTAATGT eekkdddddddddkkee 86 9833 9849 1172 TTATCA 596608 684 700 AGTGTTTAAT eekkddddddddkkeee 88 9834 9850 1173 GTTTATC 596799 684 700 AGTGTTTAAT eekkdddddddddkkee 80 9834 9850 1173 GTTTATC 596609 685 701 CAGTGTTTAAT eekkddddddddkkeee 77 9835 9851 1174 GTTTAT 596800 685 701 CAGTGTTTAAT eekkdddddddddkkee 85 9835 9851 1174 GTTTAT 596610 686 702 ACAGTGTTTA eekkddddddddkkeee 83 9836 9852 1175 ATGTTTA 596801 686 702 ACAGTGTTTA eekkdddddddddkkee 84 9836 9852 1175 ATGTTTA 596611 690 706 GATTACAGTG eekkddddddddkkeee 54 9840 9856 1179 TTTAATG 596802 690 706 GATTACAGTG eekkdddddddddkkee 61 9840 9856 1179 TTTAATG 596612 691 707 AGATTACAGT eekkddddddddkkeee 68 9841 9857 1180 GTTTAAT 596803 691 707 AGATTACAGT eekkdddddddddkkee 63 9841 9857 1180 GTTTAAT 596613 697 713 CTTTTAAGATT eekkddddddddkkeee 62 9847 9863 1353 ACAGTG 596804 697 713 CTTTTAAGATT eekkdddddddddkkee 53 9847 9863 1353 ACAGTG 596614 699 715 CACTTTTAAG eekkddddddddkkeee 37 9849 9865 1354 ATTACAG 596805 699 715 CACTTTTAAG eekkdddddddddkkee 49 9849 9865 1354 ATTACAG 596615 710 726 TCACACAATT eekkddddddddkkeee 28 9860 9876 1355 ACACTTT 596806 710 726 TCACACAATT eekkdddddddddkkee 39 9860 9876 1355 ACACTTT 596616 711 727 GTCACACAAT eekkddddddddkkeee 28 9861 9877 1356 TACACTT 596807 711 727 GTCACACAAT eekkdddddddddkkee 35 9861 9877 1356 TACACTT 596617 713 729 AAGTCACACA eekkddddddddkkeee 41 9863 9879 1357 ATTACAC 596808 713 729 AAGTCACACA eekkdddddddddkkee 37 9863 9879 1357 ATTACAC 596618 737 753 AGGTACTTTA eekkddddddddkkeee 35 9887 9903 1358 AAGCAAC 596809 737 753 AGGTACTTTA eekkdddddddddkkee 42 9887 9903 1358 AAGCAAC 596619 739 755 ACAGGTACTT eekkddddddddkkeee 14 9889 9905 1359 TAAAGCA 596810 739 755 ACAGGTACTT eekkdddddddddkkee 20 9889 9905 1359 TAAAGCA 596620 740 756 TACAGGTACT eekkddddddddkkeee 23 9890 9906 1360 TTAAAGC 596811 740 756 TACAGGTACT eekkdddddddddkkee 26 9890 9906 1360 TTAAAGC 596621 741 757 CTACAGGTAC eekkddddddddkkeee  2 9891 9907 1361 TTTAAAG 596812 741 757 CTACAGGTAC eekkdddddddddkkee 16 9891 9907 1361 TTTAAAG 596622 743 759 CACTACAGGT eekkddddddddkkeee 27 9893 9909 1362 ACTTTAA 596813 743 759 CACTACAGGT eekkdddddddddkkee 38 9893 9909 1362 ACTTTAA 596623 744 760 TCACTACAGG eekkddddddddkkeee 27 9894 9910 1363 TACTTTA 596814 744 760 TCACTACAGG eekkdddddddddkkee 35 9894 9910 1363 TACTTTA 596624 745 761 CTCACTACAG eekkddddddddkkeee 40 9895 9911 1364 GTACTTT 596815 745 761 CTCACTACAG eekkdddddddddkkee 54 9895 9911 1364 GTACTTT 596625 746 762 TCTCACTACA eekkddddddddkkeee 42 9896 9912 1365 GGTACTT 596816 746 762 TCTCACTACA eekkdddddddddkkee 46 9896 9912 1365 GGTACTT 596626 747 763 TTCTCACTACA eekkddddddddkkeee 26 9897 9913 1366 GGTACT 596817 747 763 TTCTCACTACA eekkdddddddddkkee 37 9897 9913 1366 GGTACT 596627 748 764 TTTCTCACTAC eekkddddddddkkeee 35 9898 9914 1367 AGGTAC 596818 748 764 TTTCTCACTAC eekkdddddddddkkee 45 9898 9914 1367 AGGTAC 596628 749 765 GTTTCTCACTA eekkddddddddkkeee 25 9899 9915 1368 CAGGTA 596819 749 765 GTTTCTCACTA eekkdddddddddkkee 38 9899 9915 1368 CAGGTA 596629 750 766 AGTTTCTCACT eekkddddddddkkeee 33 9900 9916 1369 ACAGGT 596820 750 766 AGTTTCTCACT eekkdddddddddkkee 50 9900 9916 1369 ACAGGT 596630 751 767 CAGTTTCTCAC eekkddddddddkkeee 38 9901 9917 1370 TACAGG 596821 751 767 CAGTTTCTCAC eekkdddddddddkkee 38 9901 9917 1370 TACAGG 596631 752 768 TCAGTTTCTCA eekkddddddddkkeee 25 9902 9918 1371 CTACAG 596822 752 768 TCAGTTTCTCA eekkdddddddddkkee 43 9902 9918 1371 CTACAG 596632 753 769 ATCAGTTTCTC eekkddddddddkkeee 31 9903 9919 1372 ACTACA 596823 753 769 ATCAGTTTCTC eekkdddddddddkkee 44 9903 9919 1372 ACTACA 596633 754 770 AATCAGTTTCT eekkddddddddkkeee 34 9904 9920 1373 CACTAC 596824 754 770 AATCAGTTTCT eekkdddddddddkkee 53 9904 9920 1373 CACTAC 596634 761 777 GATCATAAAT eekkddddddddkkeee 34 9911 9927 1374 CAGTTTC 596825 761 777 GATCATAAAT eekkdddddddddkkee 38 9911 9927 1374 CAGTTTC 596635 762 778 TGATCATAAA eekkddddddddkkeee 49 9912 9928 1375 TCAGTTT 596826 762 778 TGATCATAAA eekkdddddddddkkee 38 9912 9928 1375 TCAGTTT 596636 763 779 GTGATCATAA eekkddddddddkkeee 33 9913 9929 1376 ATCAGTT 596827 763 779 GTGATCATAA eekkdddddddddkkee 48 9913 9929 1376 ATCAGTT 596637 764 780 AGTGATCATA eekkddddddddkkeee 23 9914 9930 1377 AATCAGT 596828 764 780 AGTGATCATA eekkdddddddddkkee 32 9914 9930 1377 AATCAGT 596638 766 782 CAAGTGATCA eekkddddddddkkeee 47 9916 9932 1378 TAAATCA 596829 766 782 CAAGTGATCA eekkdddddddddkkee 29 9916 9932 1378 TAAATCA 596639 767 783 CCAAGTGATC eekkddddddddkkeee 40 9917 9933 1379 ATAAATC 596830 767 783 CCAAGTGATC eekkdddddddddkkee 48 9917 9933 1379 ATAAATC 596640 768 784 TCCAAGTGAT eekkddddddddkkeee 42 9918 9934 1380 CATAAAT 596831 768 784 TCCAAGTGAT eekkdddddddddkkee 39 9918 9934 1380 CATAAAT 596641 770 786 CTTCCAAGTG eekkddddddddkkeee 40 9920 9936 1381 ATCATAA 596832 770 786 CTTCCAAGTG eekkdddddddddkkee 54 9920 9936 1381 ATCATAA 596642 771 787 TCTTCCAAGTG eekkddddddddkkeee 33 9921 9937 1382 ATCATA 596833 771 787 TCTTCCAAGTG eekkdddddddddkkee 43 9921 9937 1382 ATCATA 596643 772 788 ATCTTCCAAGT eekkddddddddkkeee 38 9922 9938 1184 GATCAT 596834 772 788 ATCTTCCAAGT eekkdddddddddkkee 38 9922 9938 1184 GATCAT

TABLE 22  Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ ID ID SEQ SEQ NO: NO: ID ID 1 1 NO: 2 NO. 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence Chemistry inhibition Site Site NO 333611 167 186 CCGTCGCCCT eeeeeddddddddddeeeee 62 973 992 21 TCAGCACGCA 596720 167 183 TCGCCCTTCA eekkddddddddkkeee 53 973 989 966 GCACGCA 596911 167 183 TCGCCCTTCA eekkdddddddddkkee 58 973 989 966 GCACGCA 596644 773 789 AATCTTCCAA eekkddddddddkkeee 19 9923 9939 1185 GTGATCA 596835 773 789 AATCTTCCAA eekkdddddddddkkee 38 9923 9939 1185 GTGATCA 596645 774 790 AAATCTTCCA eekkddddddddkkeee 46 9924 9940 1186 AGTGATC 596836 774 790 AAATCTTCCA eekkdddddddddkkee 48 9924 9940 1186 AGTGATC 596646 782 798 AACTATACAA eekkddddddddkkeee 60 9932 9948 1193 ATCTTCC 596837 782 798 AACTATACAA eekkdddddddddkkee 63 9932 9948 1193 ATCTTCC 596647 783 799 AAACTATACA eekkddddddddkkeee 55 9933 9949 1194 AATCTTC 596838 783 799 AAACTATACA eekkdddddddddkkee 55 9933 9949 1194 AATCTTC 596648 806 822 AGACATTTTA eekkddddddddkkeee 46 9956 9972 1383 ACTGAGT 596839 806 822 AGACATTTTA eekkdddddddddkkee 53 9956 9972 1383 ACTGAGT 596649 817 833 GTCATTGAAA eekkddddddddkkeee 2 9967 9983 1201 CAGACAT 596840 817 833 GTCATTGAAA eekkdddddddddkkee 15 9967 9983 1201 CAGACAT 596650 819 835 AGGTCATTGA eekkddddddddkkeee 40 9969 9985 1203 AACAGAC 596841 819 835 AGGTCATTGA eekkdddddddddkkee 44 9969 9985 1203 AACAGAC 596651 822 838 TACAGGTCAT eekkddddddddkkeee 26 9972 9988 1206 TGAAACA 596842 822 838 TACAGGTCAT eekkdddddddddkkee 38 9972 9988 1206 TGAAACA 596652 823 839 ATACAGGTCA eekkddddddddkkeee 33 9973 9989 1207 TTGAAAC 596843 823 839 ATACAGGTCA eekkdddddddddkkee 22 9973 9989 1207 TTGAAAC 596653 825 841 AAATACAGGT eekkddddddddkkeee 28 9975 9991 1209 CATTGAA 596844 825 841 AAATACAGGT eekkdddddddddkkee 47 9975 9991 1209 CATTGAA 596654 827 843 CAAAATACAG eekkddddddddkkeee 44 9977 9993 1211 GTCATTG 596845 827 843 CAAAATACAG eekkdddddddddkkee 56 9977 9993 1211 GTCATTG 596655 830 846 TGGCAAAATA eekkddddddddkkeee 33 9980 9996 1214 CAGGTCA 596846 830 846 TGGCAAAATA eekkdddddddddkkee 43 9980 9996 1214 CAGGTCA 596656 831 847 CTGGCAAAAT eekkddddddddkkeee 25 9981 9997 1215 ACAGGTC 596847 831 847 CTGGCAAAAT eekkdddddddddkkee 53 9981 9997 1215 ACAGGTC 596657 833 849 GTCTGGCAAA eekkddddddddkkeee 30 9983 9999 1217 ATACAGG 596848 833 849 GTCTGGCAAA eekkdddddddddkkee 38 9983 9999 1217 ATACAGG 596658 836 852 TAAGTCTGGC eekkddddddddkkeee 24 9986 1ooo2 1220 AAAATAC 596849 836 852 TAAGTCTGGC eekkdddddddddkkee 46 9986 1ooo2 1220 AAAATAC 596659 837 853 TTAAGTCTGG eekkddddddddkkeee 27 9987 1ooo3 1221 CAAAATA 596850 837 853 TTAAGTCTGG eekkdddddddddkkee 42 9987 1ooo3 1221 CAAAATA 596660 840 856 GATTTAAGTC eekkddddddddkkeee 19 9990 1ooo6 1384 TGGCAAA 596851 840 856 GATTTAAGTC eekkdddddddddkkee 35 9990 1ooo6 1384 TGGCAAA 596661 841 857 TGATTTAAGT eekkddddddddkkeee 52 9991 1ooo7 1385 CTGGCAA 596852 841 857 TGATTTAAGT eekkdddddddddkkee 52 9991 1ooo7 1385 CTGGCAA 596662 842 858 GTGATTTAAG eekkddddddddkkeee 54 9992 1ooo8 1386 TCTGGCA 596853 842 858 GTGATTTAAG eekkdddddddddkkee 69 9992 1ooo8 1386 TCTGGCA 596663 843 859 TGTGATTTAA eekkddddddddkkeee 45 9993 1ooo9 1387 GTCTGGC 596854 843 859 TGTGATTTAA eekkdddddddddkkee 58 9993 1ooo9 1387 GTCTGGC 596664 844 860 CTGTGATTTA eekkddddddddkkeee n.d. 9994 10010 1388 AGTCTGG 596855 844 860 CTGTGATTTA eekkdddddddddkkee 61 9994 10010 1388 AGTCTGG 596665 845 861 TCTGTGATTT eekkddddddddkkeee 49 9995 10011 1389 AAGTCTG 596856 845 861 TCTGTGATTT eekkdddddddddkkee 49 9995 10011 1389 AAGTCTG 596666 846 862 ATCTGTGATT eekkddddddddkkeee 35 9996 10012 1390 TAAGTCT 596857 846 862 ATCTGTGATT eekkdddddddddkkee 37 9996 10012 1390 TAAGTCT 596667 847 863 CATCTGTGAT eekkddddddddkkeee 42 9997 10013 1391 TTAAGTC 596858 847 863 CATCTGTGAT eekkdddddddddkkee 48 9997 10013 1391 TTAAGTC 596668 848 864 CCATCTGTGA eekkddddddddkkeee 46 9998 10014 1392 TTTAAGT 596859 848 864 CCATCTGTGA eekkdddddddddkkee 47 9998 10014 1392 TTTAAGT 596669 849 865 CCCATCTGTG eekkddddddddkkeee 49 9999 10015 1393 ATTTAAG 596860 849 865 CCCATCTGTG eekkdddddddddkkee 49 9999 10015 1393 ATTTAAG 596670 850 866 ACCCATCTGT eekkddddddddkkeee 33 1ooo0 10016 1394 GATTTAA 596861 850 866 ACCCATCTGT eekkdddddddddkkee 44 1ooo0 10016 1394 GATTTAA 596671 851 867 TACCCATCTG eekkddddddddkkeee 29 1ooo1 10017 1395 TGATTTA 596862 851 867 TACCCATCTG eekkdddddddddkkee 45 1ooo1 10017 1395 TGATTTA 596672 854 870 TAATACCCAT eekkddddddddkkeee 25 1ooo4 10020 1223 CTGTGAT 596863 854 870 TAATACCCAT eekkdddddddddkkee 28 1ooo4 10020 1223 CTGTGAT 596673 855 871 TTAATACCCA eekkddddddddkkeee 28 1ooo5 10021 1224 TCTGTGA 596864 855 871 TTAATACCCA eekkdddddddddkkee 26 1ooo5 10021 1224 TCTGTGA 596674 858 874 AGTTTAATAC eekkddddddddkkeee 29 1ooo8 10024 1227 CCATCTG 596865 858 874 AGTTTAATAC eekkdddddddddkkee 43 1ooo8 10024 1227 CCATCTG 596675 859 875 AAGTTTAATA eekkddddddddkkeee 54 1ooo9 10025 1228 CCCATCT 596866 859 875 AAGTTTAATA eekkdddddddddkkee 59 1ooo9 10025 1228 CCCATCT 596676 860 876 CAAGTTTAAT eekkddddddddkkeee 52 10010 10026 1229 ACCCATC 596867 860 876 CAAGTTTAAT eekkdddddddddkkee 62 10010 10026 1229 ACCCATC 596677 861 877 ACAAGTTTAA eekkddddddddkkeee 58 10011 10027 1230 TACCCAT 596868 861 877 ACAAGTTTAA eekkdddddddddkkee 61 10011 10027 1230 TACCCAT 596678 862 878 GACAAGTTTA eekkddddddddkkeee 54 10012 10028 1231 ATACCCA 596869 862 878 GACAAGTTTA eekkdddddddddkkee 59 10012 10028 1231 ATACCCA 596679 863 879 TGACAAGTTT eekkddddddddkkeee 43 10013 10029 1232 AATACCC 596870 863 879 TGACAAGTTT eekkdddddddddkkee 52 10013 10029 1232 AATACCC 596680 864 880 CTGACAAGTT eekkddddddddkkeee 30 10014 10030 1233 TAATACC 596871 864 880 CTGACAAGTT eekkdddddddddkkee 36 10014 10030 1233 TAATACC 596681 865 881 TCTGACAAGT eekkddddddddkkeee 33 10015 10031 1234 TTAATAC 596872 865 881 TCTGACAAGT eekkdddddddddkkee 20 10015 10031 1234 TTAATAC

TABLE 23  Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ ID ID SEQ SEQ NO: NO: ID ID 1 1 NO. 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequence Chemistry inhibition Site Site NO 333611 167 186 CCGTCGCCCT eeeeeddddddddddeeeee 68 973 992 21 TCAGCACGCA 596720 167 183 TCGCCCTTCA eekkddddddddkkeee 64 973 989 966 GCACGCA 596911 167 183 TCGCCCTTCA eekkdddddddddkkee 71 973 989 966 GCACGCA 596682 884 900 GCTTGAATGA eekkddddddddkkeee 24 10034 10050 1396 CAAAGAA 596873 884 900 GCTTGAATGA eekkdddddddddkkee 32 10034 10050 1396 CAAAGAA 596683 885 901 GGCTTGAATG eekkddddddddkkeee 54 10035 10051 1397 ACAAAGA 596874 885 901 GGCTTGAATG eekkdddddddddkkee 44 10035 10051 1397 ACAAAGA 596684 889 905 CACAGGCTTG eekkddddddddkkeee 34 10039 10055 1398 AATGACA 596875 889 905 CACAGGCTTG eekkdddddddddkkee 47 10039 10055 1398 AATGACA 596685 890 906 TCACAGGCTT eekkddddddddkkeee 28 10040 10056 1399 GAATGAC 596876 890 906 TCACAGGCTT eekkdddddddddkkee 46 10040 10056 1399 GAATGAC 596686 891 907 TTCACAGGCT eekkddddddddkkeee 20 10041 10057 1242 TGAATGA 596877 891 907 TTCACAGGCT eekkdddddddddkkee 16 10041 10057 1242 TGAATGA 596687 892 908 ATTCACAGGC eekkddddddddkkeee 19 10042 10058 1243 TTGAATG 596878 892 908 ATTCACAGGC eekkdddddddddkkee 29 10042 10058 1243 TTGAATG 596688 893 909 TATTCACAGG eekkddddddddkkeee 24 10043 10059 1244 CTTGAAT 596879 893 909 TATTCACAGG eekkdddddddddkkee 11 10043 10059 1244 CTTGAAT 596689 894 910 TTATTCACAG eekkddddddddkkeee 26 10044 10060 1245 GCTTGAA 596880 894 910 TTATTCACAG eekkdddddddddkkee 30 10044 10060 1245 GCTTGAA 596690 895 911 TTTATTCACA eekkddddddddkkeee 44 10045 10061 1246 GGCTTGA 596881 895 911 TTTATTCACA eekkdddddddddkkee 55 10045 10061 1246 GGCTTGA 596691 896 912 TTTTATTCAC eekkddddddddkkeee 43 10046 10062 1247 AGGCTTG 596882 896 912 TTTTATTCAC eekkdddddddddkkee 48 10046 10062 1247 AGGCTTG 596692 899 915 GGTTTTTATT eekkddddddddkkeee 38 10049 10065 1250 CACAGGC 596883 899 915 GGTTTTTATT eekkdddddddddkkee 57 10049 10065 1250 CACAGGC 596693 903 919 ACAGGGTTTT eekkddddddddkkeee 29 10053 10069 1254 TATTCAC 596884 903 919 ACAGGGTTTT eekkdddddddddkkee 47 10053 10069 1254 TATTCAC 596694 904 920 TACAGGGTTT eekkddddddddkkeee 13 10054 10070 1255 TTATTCA 596885 904 920 TACAGGGTTT eekkdddddddddkkee 31 10054 10070 1255 TTATTCA 596695 907 923 CCATACAGGG eekkddddddddkkeee 13 10057 10073 1258 TTTTTAT 596886 907 923 CCATACAGGG eekkdddddddddkkee 34 10057 10073 1258 TTTTTAT 596696 908 924 GCCATACAGG eekkddddddddkkeee 13 10058 10074 1259 GTTTTTA 596887 908 924 GCCATACAGG eekkdddddddddkkee 26 10058 10074 1259 GTTTTTA 596697 909 925 TGCCATACAG eekkddddddddkkeee 21 10059 10075 1260 GGTTTTT 596888 909 925 TGCCATACAG eekkdddddddddkkee 22 10059 10075 1260 GGTTTTT 596698 910 926 GTGCCATACA eekkddddddddkkeee 20 10060 10076 1261 GGGTTTT 596889 910 926 GTGCCATACA eekkdddddddddkkee 28 10060 10076 1261 GGGTTTT 596699 911 927 AGTGCCATAC eekkddddddddkkeee 20 10061 10077 1262 AGGGTTT 596890 911 927 AGTGCCATAC eekkdddddddddkkee 27 10061 10077 1262 AGGGTTT 596700 912 928 AAGTGCCATA eekkddddddddkkeee 15 10062 10078 1400 CAGGGTT 596891 912 928 AAGTGCCATA eekkdddddddddkkee 21 10062 10078 1400 CAGGGTT 596701 913 929 TAAGTGCCAT eekkddddddddkkeee 26 10063 10079 1401 ACAGGGT 596892 913 929 TAAGTGCCAT eekkdddddddddkkee 35 10063 10079 1401 ACAGGGT 596702 914 930 ATAAGTGCCA eekkddddddddkkeee 36 10064 10080 1402 TACAGGG 596893 914 930 ATAAGTGCCA eekkdddddddddkkee 46 10064 10080 1402 TACAGGG 596703 915 931 AATAAGTGCC eekkddddddddkkeee 40 10065 10081 1403 ATACAGG 596894 915 931 AATAAGTGCC eekkdddddddddkkee 36 10065 10081 1403 ATACAGG 596704 916 932 TAATAAGTGC eekkddddddddkkeee 22 10066 10082 1404 CATACAG 596895 916 932 TAATAAGTGC eekkdddddddddkkee 30 10066 10082 1404 CATACAG 596705 917 933 ATAATAAGTG eekkddddddddkkeee 27 10067 10083 1405 CCATACA 596896 917 933 ATAATAAGTG eekkdddddddddkkee 27 10067 10083 1405 CCATACA 596706 918 934 CATAATAAGT eekkddddddddkkeee 32 10068 10084 1406 GCCATAC 596897 918 934 CATAATAAGT eekkdddddddddkkee 34 10068 10084 1406 GCCATAC 596707 919 935 TCATAATAAG eekkddddddddkkeee 28 10069 10085 1407 TGCCATA 596898 919 935 TCATAATAAG eekkdddddddddkkee 34 10069 10085 1407 TGCCATA 596708 920 936 CTCATAATAA eekkddddddddkkeee 30 10070 10086 1408 GTGCCAT 596899 920 936 CTCATAATAA eekkdddddddddkkee 44 10070 10086 1408 GTGCCAT 596709 921 937 CCTCATAATA eekkddddddddkkeee 29 10071 10087 1409 AGTGCCA 596900 921 937 CCTCATAATA eekkdddddddddkkee 31 10071 10087 1409 AGTGCCA 596710 922 938 GCCTCATAAT eekkddddddddkkeee 41 10072 10088 1410 AAGTGCC 596901 922 938 GCCTCATAAT eekkdddddddddkkee 33 10072 10088 1410 AAGTGCC 596711 923 939 AGCCTCATAA eekkddddddddkkeee 16 10073 10089 1411 TAAGTGC 596902 923 939 AGCCTCATAA eekkdddddddddkkee 11 10073 10089 1411 TAAGTGC 596712 924 940 TAGCCTCATA eekkddddddddkkeee 11 10074 10090 1412 ATAAGTG 596903 924 940 TAGCCTCATA eekkdddddddddkkee 27 10074 10090 1412 ATAAGTG 596713 925 941 ATAGCCTCAT eekkddddddddkkeee 20 10075 10091 1413 AATAAGT 596904 925 941 ATAGCCTCAT eekkdddddddddkkee 27 10075 10091 1413 AATAAGT 596714 926 942 AATAGCCTCA eekkddddddddkkeee 20 10076 10092 1414 TAATAAG 596905 926 942 AATAGCCTCA eekkdddddddddkkee 25 10076 10092 1414 TAATAAG 596715 931 947 CTTTTAATAG eekkddddddddkkeee 45 10081 10097 1415 CCTCATA 596906 931 947 CTTTTAATAG eekkdddddddddkkee 34 10081 10097 1415 CCTCATA 596716 932 948 TCTTTTAATA eekkddddddddkkeee 52 10082 10098 1416 GCCTCAT 596907 932 948 TCTTTTAATA eekkdddddddddkkee 56 10082 10098 1416 GCCTCAT 596717 936 952 GGATTCTTTT eekkddddddddkkeee 14 10086 10102 1417 AATAGCC 596908 936 952 GGATTCTTTT eekkdddddddddkkee 19 10086 10102 1417 AATAGCC 596718 938 954 TTGGATTCTT eekkddddddddkkeee 23 10088 10104 1263 TTAATAG 596909 938 954 TTGGATTCTT eekkdddddddddkkee 8 10088 10104 1263 TTAATAG 596719 949 965 TTAGTTTGAA eekkddddddddkkeee 31 10099 10115 1418 TTTGGAT 596910 949 965 TTAGTTTGAA eekkdddddddddkkee 16 10099 10115 1418 TTTGGAT

Example 6: Dose-Dependent Inhibition of Human SOD-1 with Modified Oligonucleotides in HepG2 Cells

Gapmers from the studies described above exhibiting significant in vitro inhibition of SOD-1 mRNA were selected and tested at various doses in HepG2 cells. Benchmark compound ISIS 333611 and other compounds previously disclosed in WO 2005/040180, including ISIS 146144, 146145, 150445, 150446, 150447, 150454, 150463, 150465, 333606, 333609, and 333611 were also tested.

The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.813 μM, 1.625 μM, 3.250 μM, 6.500 μM, and 13.000 μM concentrations of modified oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3898 was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. SOD-1 mRNA levels were significantly reduced in a dose-dependent manner in modified oligonucleotide treated cells.

TABLE 24 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.813 1.625 3.250 6.500 13.000 IC₅₀ No μM μM μM μM μM (μM) 150445 7 21 44 56 82 4.4 150446 15 32 47 71 87 3.2 150447 26 43 68 81 93 2.0 150463 16 38 51 66 85 3.1 333611 18 39 57 66 79 3.0 393336 18 34 53 69 83 3.1 393343 24 32 53 73 42 5.1 436863 18 42 58 72 89 2.6 590089 28 59 70 82 90 1.5 590090 34 56 76 82 51 1.1 590091 30 44 68 84 88 1.9 590094 16 35 57 73 76 3.0 590113 34 35 57 73 84 2.3

TABLE 25 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.813 1.625 3.250 6.500 13.000 IC₅₀ No μM μM μM μM μM (μM) 150465 42 59 77 82 87 1.0 333611 17 26 40 64 82 3.8 378879 14 35 63 72 86 2.8 393371 28 42 57 74 80 2.3 489520 28 44 64 72 84 2.2 590177 53 59 69 85 88 0.7 590178 40 53 71 73 87 1.3 590180 18 42 51 64 73 3.3 590187 34 51 68 80 92 1.6 590188 30 46 61 76 88 2.0 590189 37 49 68 78 88 1.6 590190 38 58 77 84 89 1.1 590191 29 56 71 77 84 1.6 590192 37 59 72 80 87 1.2

TABLE 26 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.813 1.625 3.250 6.500 13.000 IC₅₀ No μM μM μM μM μM (μM) 146144 15 58 67 78 64 2.2 146145 31 53 67 81 90 1.6 333606 11 39 62 75 91 2.7 333609 13 37 57 71 85 2.9 333611 14 30 53 68 86 3.2 590250 8 19 47 64 84 3.9 590626 61 72 84 84 87 0.2 592630 24 33 58 70 85 2.7 592631 19 48 59 74 88 2.3 592645 20 31 53 74 89 2.8 592649 14 32 56 69 82 3.2

TABLE 27 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.813 1.625 3.250 6.500 13.000 IC₅₀ No μM μM μM μM μM (μM) 150454 13 24 49 69 83 3.5 333611 28 28 53 68 82 3.0 489505 13 24 38 56 81 4.5 489516 25 16 39 61 79 4.3 592652 11 31 52 74 46 5.1 592714 8 25 45 69 82 3.8 592715 18 35 50 70 83 3.1 592762 44 66 74 81 89 0.8 592763 50 68 74 86 95 0.7 592764 26 43 48 76 87 2.5 592766 36 53 66 77 89 1.5 592767 25 31 54 70 79 2.9 592769 35 31 56 73 80 2.5 592771 34 43 58 70 80 2.2

Example 7: Dose-Dependent Inhibition of Human SOD-1 with Modified Oligonucleotides in HepG2 Cells

Gapmers from the studies described above exhibiting significant in vitro inhibition of SOD-1 mRNA were selected and tested at various doses in HepG2 cells. Benchmark compound, ISIS 333611, and ISIS 333625, both of which were previously disclosed in WO 2005/040180 were also tested.

The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.148 μM, 0.444 μM, 1.330 μM, 4.000 μM, and 12.000 μM concentrations of modified oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR. Human primer probe sets RTS3898 or HTS90 was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. SOD-1 mRNA levels were significantly reduced in a dose-dependent manner in modified oligonucleotide treated cells.

TABLE 28 Dose response assay with primer probe set RTS3898 ISIS 0.148 0.444 1.330 4.000 12.000 IC₅₀ No μM μM μM μM μM (μM) 333611 6 14 29 51 78 3.2 596911 8 12 23 54 74 3.6 596720 7 14 31 55 71 3.4 596800 44 60 75 84 82 0.2 596801 33 49 69 79 83 0.5 596610 16 44 65 78 84 0.8 596799 20 45 64 75 84 0.8 596609 17 54 65 75 81 0.7 596883 13 22 36 45 51 8.6 489523 16 40 62 78 90 0.9 590181 5 17 46 70 89 1.7 436868 10 35 47 69 82 1.4 596768 16 37 62 82 89 0.9 596775 36 50 66 83 89 0.4

TABLE 29 Dose response assay with primer probe set HTS90 ISIS 0.148 0.444 1.330 4.000 12.000 IC₅₀ No μM μM μM μM μM (μM) 333625 0 4 17 56 84 3.3 489532 54 70 78 86 96 0.1 590154 0 14 25 56 86 2.7 596173 0 5 25 63 92 2.4 596174 7 12 37 46 84 2.8 596178 2 16 40 68 82 2.1 596179 0 17 41 64 80 2.3 596308 0 5 22 56 80 3.3 596572 18 35 62 83 90 0.9 596589 10 45 61 77 91 0.9 596600 41 56 71 85 92 0.3 596602 14 51 74 86 95 0.6 596789 22 55 69 86 91 0.5 596795 16 43 64 82 93 0.8

Example 8: Dose-Dependent Inhibition of Human SOD-1 with Modified Oligonucleotides in HepG2 Cells

Gapmers from the studies described above exhibiting significant in vitro inhibition of SOD-1 mRNA were selected and tested at various doses in HepG2 cells. Benchmark compound, ISIS 333611, and additional compounds including, ISIS 146143, 150442, 195753, 333607, and 333608, were previously disclosed in WO 2005/040180 were also tested.

The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.1875 μM, 0.7500 μM, 3.0000 μM, and 12.0000 μM concentrations of modified oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR. Human primer probe sets RTS3898 was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells. The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. SOD-1 mRNA levels were significantly reduced in a dose-dependent manner in modified oligonucleotide treated cells.

TABLE 30 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.1875 0.75 3.00 12.00 IC₅₀ No μM μM μM μM (μM) 333611 0 27 60 91 2 596572 38 65 87 96 0.3 596583 62 89 95 95 <0.1 596590 40 79 89 94 0.2 596602 40 75 92 98 0.2 596603 51 79 92 96 0.1 596604 48 78 91 95 0.1 596605 50 86 90 97 0.1 596764 11 67 89 94 0.7 596768 27 49 82 92 0.7 596773 32 62 89 98 0.4 596774 56 89 93 96 <0.1 596775 31 75 90 97 0.3 596780 24 71 85 98 0.5 596781 30 80 93 97 0.3 596791 38 74 89 95 0.3 596794 43 75 91 97 0.2 596795 28 66 91 98 0.4 596796 43 78 93 98 0.2

TABLE 31 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.1875 0.75 3.00 12.00 IC₅₀ No μM μM μM μM (μM) 333611 0 15 68 95 2.1 596589 35 70 90 97 0.3 596789 38 71 89 97 0.3 596600 41 73 89 95 0.2 596582 30 71 87 95 0.4 596784 26 68 89 95 0.4 596787 44 67 89 94 0.2 596779 29 71 89 97 0.4 596792 37 63 83 95 0.4 596782 27 61 85 96 0.5 596765 34 59 87 95 0.4 596793 27 65 88 96 0.5 596570 25 60 84 91 0.6 596769 21 64 85 96 0.6 596783 10 57 84 94 0.9 596584 26 67 84 93 0.5 596571 37 71 81 92 0.3 596598 30 62 81 94 0.5 596588 11 64 87 95 0.7

TABLE 32 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.1875 0.75 3.00 12.00 IC₅₀ No μM μM μM μM (μM) 146143 6 12 51 88 2.5 150442 10 21 39 90 2.5 195753 13 23 48 77 2.8 333607 17 26 59 83 1.9 333608 0 2 28 92 3.7 333611 0 13 52 91 2.4 596573 26 51 77 91 0.8 596577 32 55 78 93 0.6 596591 23 51 74 91 0.8 596592 18 48 66 86 1.1 596593 16 58 78 87 0.8 596596 4 49 72 87 1.3 596761 28 55 74 91 0.7 596762 0 47 75 90 1.3 596763 0 40 78 92 1.5 596766 4 50 68 86 1.3 596785 10 47 77 91 1.1 596786 0 45 75 97 1.3 596788 9 52 81 95 1

TABLE 33 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.1875 0.75 3.00 12.00 IC₅₀ No μM μM μM μM (μM) 333611 3 22 60 92 2 596302 9 27 59 89 1.9 596308 29 47 82 96 0.7 596309 13 42 75 92 1.1 596310 13 16 48 81 2.8 596313 15 37 70 88 1.3 596314 18 45 74 92 1 596606 55 78 87 93 0.1 596607 44 71 83 84 0.2 596608 46 74 84 81 0.1 596609 30 61 79 87 0.5 596610 39 69 82 86 0.3 596612 16 50 62 77 1.4 596797 67 84 94 96 <0.1 596798 42 68 86 89 0.2 596799 35 66 83 87 0.4 596800 45 73 84 87 0.2 596801 40 67 86 88 0.3 596803 28 41 63 71 1.4

TABLE 34 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.1875 0.75 3.00 12.00 IC₅₀ No μM μM μM μM (μM) 333611 0 30 56 69 3.1 590475 19 39 69 88 1.2 590625 19 51 74 85 1.0 590626 42 72 88 90 0.2 590627 16 42 70 84 1.0 590634 45 72 86 92 0.2 590635 39 60 80 90 0.4 590644 44 62 80 86 0.3 590650 34 56 82 93 0.5 590653 52 78 86 85 0.1 590655 35 71 79 82 0.3 596530 25 22 72 79 2.0 596531 8 38 74 96 1.2 596559 15 36 79 95 1.1 596721 14 47 82 97 0.9 596723 12 47 79 94 1.0 596726 24 42 80 94 0.9 596735 7 32 77 96 1.3 596736 25 52 82 97 0.7

Example 9: Dose-Dependent Inhibition of Human SOD-1 in HepG2 Cells by Gapmers with Mixed Backbone Chemistry

Additional gapmers were designed based on the sequences of the oligonucleotides disclosed in studies described above. The oligonucleotides were designed as 5-10-5 MOE, 5-8-5 MOE, and deoxy, MOE and cEt oligonucleotides. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment is comprised of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment is comprised of eight 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The deoxy, MOE and cEt oligonucleotides are 16 or 17 nucleosides in length wherein each nucleoside has a MOE sugar modification, a cEt sugar modification, or a deoxy moiety. The sugar chemistry of each oligonucleotide is denoted as in the Chemistry column, where ‘k’ indicates a cEt modified sugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’ indicates a 2′-MOE modified sugar. The internucleoside linkages throughout each gapmer are either phosphodiester or phosphorothioate linkages. The internucleoside linkages of each oligonucleotide are denoted in the Backbone Chemistry column, where ‘o’ indicates a phosphodiester linkage and ‘s’ denotes a phosphorothioate linkage. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human SOD-1 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the human SOD-1 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000 to 18704000).

TABLE 35  Modified oligonucleotides targeting human SOD-1 with mixed backbone chemistry SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: NO: 1 1 2 2 SEQ ISIS Start Stop Backbone Start Stop ID NO Site Site Sequence Sugar Modifications Chemistry Site Site NO 611458 226 245 ACACCTTCAC eeeeeddddddddddeeeee sooosssssssssssooos 4980 4999 23 TGGTCCATTA 654335 233 248 CCCACACCTT ekddddddddekekee sossssssssoooss 4987 5002 1420 CACTGG 611474 234 253 GCTTCCCCAC eeeeeddddddddddeeeee sooosssssssssssooos 4988 5007 149 ACCTTCACTG 654301 234 251 TTCCCCACAC eeeeeddddddddeeeee sooosssssssssooss 4988 5005 1421 CTTCACTG 654336 234 249 CCCCACACCT ekddddddddekekee sossssssssoooss 4988 5003 1422 TCACTG 654302 235 252 CTTCCCCACA eeeeeddddddddeeeee sooosssssssssooss 4989 5006 1423 CCTTCACT 654319 235 250 TCCCCACACC kekeddddddddekek sooossssssssoss 4989 5004 1424 TTCACT 654337 235 250 TCCCCACACC ekddddddddekekee sossssssssoooss 4989 5004 1424 TTCACT 654303 236 253 GCTTCCCCAC eeeeeddddddddeeeee sooosssssssssooss 4990 5007 1425 ACCTTCAC 654320 236 251 TTCCCCACAC kekeddddddddekek sooossssssssoss 4990 5005 1426 CTTCAC 654321 237 252 CTTCCCCACA kekeddddddddekek sooossssssssoss 4991 5006 1427 CCTTCA 611475 321 340 GTGAGGACCT eeeeeddddddddddeeeee sooosssssssssssooos 7637 7656 172 GCACTGGTAC 611460 588 607 GGCGATCCCA eeeeeddddddddddeeeee sooosssssssssssooos 9738 9757 47 ATTACACCAC 654304 663 680 ATACATTTCT eeeeeddddddddeeeee sooosssssssssooss 9813 9830 1429 ACAGCTAG 654305 664 681 GATACATTTC eeeeeddddddddeeeee sooosssssssssooss 9814 9831 1430 TACAGCTA 654340 664 679 TACATTTCTA ekddddddddekekee sossssssssoooss 9814 9829 1431 CAGCTA 611492 665 684 CAGGATACAT eeeeeddddddddddeeeee sooosssssssssssooos 9815 9834 725 TTCTACAGCT 654306 665 682 GGATACATTT eeeeeddddddddeeeee sooosssssssssooss 9815 9832 1432 CTACAGCT 654323 665 680 ATACATTTCT kekeddddddddekek sooossssssssoss 9815 9830 1433 ACAGCT 654341 665 680 ATACATTTCT ekddddddddekekee sossssssssoooss 9815 9830 1433 ACAGCT 611500 666 685 TCAGGATACA eeeeeddddddddddeeeee sooosssssssssssooos 9816 9835 823 TTTCTACAGC 612941 666 682 GGATACATTT eekkdddddddddkkee sooosssssssssoss 9816 9832 1342 CTACAGC 654307 666 683 AGGATACATT eeeeeddddddddeeeee sooosssssssssooss 9816 9833 1434 TCTACAGC 654324 666 681 GATACATTTC kekeddddddddekek sooossssssssoss 9816 9831 1435 TACAGC 654342 666 681 GATACATTTC ekddddddddekekee sossssssssoooss 9816 9831 1435 TACAGC 654308 667 684 CAGGATACAT eeeeeddddddddeeeee sooosssssssssooss 9817 9834 1436 TTCTACAG 612925 676 692 ATGTTTATCA eekkddddddddkkeee soosssssssssooss 9826 9842 1348 GGATACA 612944 676 692 ATGTTTATCA eekkdddddddddkkee sooosssssssssoss 9826 9842 1348 GGATACA 654343 677 692 ATGTTTATCA ekddddddddekekee sossssssssoooss 9827 9842 1437 GGATAC 612927 678 694 TAATGTTTAT eekkddddddddkkeee soosssssssssooss 9828 9844 1350 CAGGATA 654309 678 695 TTAATGTTTA eeeeeddddddddeeeee sooosssssssssooss 9828 9845 1438 TCAGGATA 612928 679 695 TTAATGTTTA eekkddddddddkkeee soosssssssssooss 9829 9845 1351 TCAGGAT 654310 679 696 TTTAATGTTT eeeeeddddddddeeeee sooosssssssssooss 9829 9846 1439 ATCAGGAT 654311 680 697 GTTTAATGTT eeeeeddddddddeeeee sooosssssssssooss 9830 9847 1440 TATCAGGA 654327 680 695 TTAATGTTTA kekeddddddddekek sooossssssssoss 9830 9845 1441 TCAGGA 654346 680 695 TTAATGTTTA ekddddddddekekee sossssssssoooss 9830 9845 1441 TCAGGA 611497 681 700 AGTGTTTAAT eeeeeddddddddddeeeee sooosssssssssssooos 9831 9850 733 GTTTATCAGG 612948 681 697 GTTTAATGTT eekkdddddddddkkee sooosssssssssoss 9831 9847 1352 TATCAGG 654312 681 698 TGTTTAATGT eeeeeddddddddeeeee sooosssssssssooss 9831 9848 1442 TTATCAGG 654328 681 696 TTTAATGTTT kekeddddddddekek sooossssssssoss 9831 9846 1443 ATCAGG 654347 681 696 TTTAATGTTT ekddddddddekekee sossssssssoooss 9831 9846 1443 ATCAGG 654313 682 699 GTGTTTAATG eeeeeddddddddeeeee sooosssssssssooss 9832 9849 1444 TTTATCAG 654329 682 697 GTTTAATGTT kekeddddddddekek sooossssssssoss 9832 9847 1445 TATCAG 654348 682 697 GTTTAATGTT ekddddddddekekee sossssssssoooss 9832 9847 1445 TATCAG 612949 683 699 GTGTTTAATG eekkdddddddddkkee sooosssssssssoss  9833 9849 1172 TTTATCA 654314 683 700 AGTGTTTAAT eeeeeddddddddeeeee sooosssssssssooss  9833 9850 1446 GTTTATCA 654330 683 698 TGTTTAATGT kekeddddddddekek sooossssssssoss 9833 9848 1447 TTATCA 612931 684 700 AGTGTTTAAT eekkddddddddkkeee soosssssssssooss  9834 9850 1173 GTTTATC 654315 684 701 CAGTGTTTAA eeeeeddddddddeeeee sooosssssssssooss  9834 9851 1448 TGTTTATC 654331 684 699 GTGTTTAATG kekeddddddddekek sooossssssssoss 9834 9849 1449 TTTATC 654350 684 699 GTGTTTAATG ekddddddddekekee sossssssssoooss 9834 9849 1449 TTTATC 654316 685 702 ACAGTGTTTA eeeeeddddddddeeeee sooosssssssssooss  9835 9852 1450 ATGTTTAT 612918 686 702 ACAGTGTTTA eeekkdddddddkkeee soosssssssssooss  9836 9852 1175 ATGTTTA 612932 686 702 ACAGTGTTTA eekkddddddddkkeee soosssssssssooss  9836 9852 1175 ATGTTTA 654317 686 703 TACAGTGTTT eeeeeddddddddeeeee sooosssssssssooss  9836 9853 1451 AATGTTTA 654333 686 701 CAGTGTTTAA kekeddddddddekek sooossssssssoss 9836 9851 1452 TGTTTA 654352 686 701 CAGTGTTTAA ekddddddddekekee sossssssssoooss 9836 9851 1452 TGTTTA 654318 687 704 TTACAGTGTT eeeeeddddddddeeeee sooosssssssssooss  9837 9854 1453 TAATGTTT 654334 687 702 ACAGTGTTTA kekeddddddddekek sooossssssssoss 9837 9852 1454 ATGTTT

The newly designed oligonucleotides were tested at various doses in HepG2 cells. The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.222 μM, 0.667 μM, 2.000 μM, and 6.000 μM concentrations of modified oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR. Human primer probe sets RTS3898 was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. SOD-1 mRNA levels were significantly reduced in a dose-dependent manner in modified oligonucleotide treated cells.

TABLE 36 Dose response assay ISIS 0.222 0.667 2.000 6.000 IC₅₀ No μM μM μM μM μM 333611 0 28 53 77 1.9 611458 0 21 50 79 2.0 611460 24 34 55 79 1.3 611474 14 32 55 79 1.5 611475 0 16 35 70 3.0 611492 38 70 88 95 0.3 611497 28 55 80 89 0.6 611500 25 50 73 92 0.7 612918 51 70 74 80 <0.2 612925 53 73 90 89 <0.2 612927 64 89 92 94 <0.2 612928 67 90 94 97 <0.2 612931 68 76 84 86 <0.2 612932 61 73 88 91 <0.2 612941 62 78 91 95 <0.2 612944 47 71 82 92 0.2 612948 76 90 93 94 <0.2 612949 58 68 83 96 <0.2 654301 7 4 17 42 >6.0

TABLE 37 Dose response assay ISIS 0.222 0.667 2.000 6.000 IC₅₀ No μM μM μM μM μM 333611 14 20 35 69 3.0 611458 11 27 36 68 2.9 654302 0 8 38 48 6.2 654303 8 29 46 76 1.9 654304 7 28 54 79 1.7 654305 28 59 73 85 0.6 654306 38 62 82 94 0.4 654307 9 43 65 86 1.1 654308 14 31 54 84 1.4 654309 0 17 47 72 2.4 654310 10 24 28 53 6.6 654311 45 73 78 87 0.2 654312 14 39 59 77 1.3 654313 20 43 56 81 1.2 654314 33 58 74 86 0.5 654315 21 47 64 84 0.9 654316 19 30 46 70 2.0 654317 13 46 57 70 1.4 654318 17 42 54 76 1.4

TABLE 38 Dose response assay ISIS 0.222 0.667 2.000 6.000 IC₅₀ No μM μM μM μM μM 333611 14 19 50 73 2.1 611458 9 22 39 72 2.5 654319 19 9 31 61 5.1 654320 6 16 20 59 5.9 654321 8 14 51 76 2.1 654323 55 73 89 95 <0.2 654324 54 78 89 96 <0.2 654327 53 82 91 96 <0.2 654328 73 90 93 97 <0.2 654329 58 78 86 94 <0.2 654330 42 54 69 86 0.4 654331 53 78 82 90 <0.2 654333 50 67 81 86 0.2 654334 55 68 78 88 <0.2 654335 15 31 58 81 1.4 654336 21 36 60 75 1.3 654337 16 34 58 80 1.4 654340 36 69 83 95 0.4 654341 43 58 79 91 0.3

TABLE 39 Dose response assay ISIS 0.222 0.667 2.00 6.00 IC₅₀ No μM μM μM μM μM 333611 0 6 38 64 3.6 611458 3 14 36 63 3.6 654342 40 60 80 93 0.4 654343 64 81 90 94 <0.2 654346 52 73 84 93 <0.2 654347 21 38 58 81 1.2 654348 44 63 82 94 0.3 654350 40 63 76 86 0.3 654352 54 79 84 88 <0.2

Example 10: Dose-Dependent Inhibition of Human SOD-1 by Gapmers with Mixed Backbone Chemistry

Additional gapmers were designed based on the sequences of the oligonucleotides disclosed in studies described above. The oligonucleotides were designed as deoxy, MOE and cEt oligonucleotides. The deoxy, MOE and cEt oligonucleotides are 16 or 17 nucleosides in length wherein each nucleoside has a MOE sugar modification, a cEt sugar modification, or a deoxy moiety. The sugar chemistry of each oligonucleotide is denoted as in the Chemistry column, where ‘k’ indicates a cEt modified sugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’ indicates a 2′-MOE modified sugar. The internucleoside linkages throughout each gapmer are either phosphodiester or phosphorothioate linkages. The internucleoside linkages of each oligonucleotide are denoted in the Backbone Chemistry column, where ‘o’ indicates a phosphodiester linkage and ‘s’ indicates a phosphorothioate linkage. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human SOD-1 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the human SOD-1 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000 to 18704000).

TABLE 40  Modified oligonucleotides targeting human SOD-1 with mixed backbone chemistry SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: NO 1 1 2 2 SEQ ISIS Start Stop Backbone Start Stop ID NO Site Site Sequence Sugar Modifications Chemistry Site Site NO 612916 664 680 ATACATTTCTA eeekkdddddddkkeee soosssssssssooss 9814 9830 1170 CAGCTA 612947 679 695 TTAATGTTTAT eekkdddddddddkkee sooosssssssssoss 9829 9845 1351 CAGGAT 654322 664 679 TACATTTCTAC kekeddddddddekek sooossssssssoss  9814 9829 1431 AGCTA 654325 667 682 GGATACATTT kekeddddddddekek sooossssssssoss  9817 9832 1455 CTACAG 654326 679 694 TAATGTTTATC kekeddddddddekek sooossssssssoss  9829 9844 1456 AGGAT 654332 685 700 AGTGTTTAAT kekeddddddddekek sooossssssssoss  9835 9850 1457 GTTTAT 654338 662 677 CATTTCTACAG ekddddddddekekee sossssssssoooss  9812 9827 1458 CTAGC 654339 663 678 ACATTTCTACA ekddddddddekekee sossssssssoooss  9813 9828 1459 GCTAG 654344 678 693 AATGTTTATCA ekddddddddekekee sossssssssoooss  9828 9843 1460 GGATA 654345 679 694 TAATGTTTATC ekddddddddekekee sossssssssoooss  9829 9844 1456 AGGAT 654349 683 698 TGTTTAATGTT ekddddddddekekee sossssssssoooss  9833 9848 1447 TATCA 654351 685 700 AGTGTTTAAT ekddddddddekekee sossssssssoooss  9835 9850 1457 GTTTAT

The newly designed oligonucleotides were tested at various doses in A431 cells. The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 5,000 cells per well and modified oligonucleotides were added to the media at 0.12 μM, 0.60 μM, 3.00 μM, and 15.00 μM concentrations of modified oligonucleotide for free uptake by the cells, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR. Human primer probe sets RTS3898 was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells.

TABLE 41 Dose response assay ISIS 0.12 0.60 3.00 15.00 No μM μM μM μM 333611 0 7 17 31 611458 5 4 4 10 612916 4 13 26 37 612918 12 26 45 47 612944 1 4 21 29 612947 12 48 54 72 654305 7 15 39 52 654306 0 25 29 50 654313 1 15 36 50 654314 2 35 52 67 654321 0 8 8 18 654322 0 13 36 59 654329 7 7 41 66 654330 6 14 15 32 654337 0 0 7 21 654338 3 3 2 11 654345 1 9 22 46 654346 0 7 21 46

TABLE 42 Dose response assay ISIS 0.12 0.60 3.00 15.00 No μM μM μM μM 333611 0 0 0 2 611460 0 0 0 30 611474 0 0 11 0 612925 2 4 12 52 612927 0 38 54 68 612948 25 69 89 95 612949 22 57 73 84 654307 42 23 26 45 654308 2 31 9 18 654315 0 8 39 52 654316 0 18 26 45 654323 15 14 16 52 654324 12 22 21 34 654331 7 35 66 78 654332 2 31 47 61 654339 1 27 32 47 654340 37 0 22 12 654347 20 5 12 33 654348 2 19 33 62

TABLE 43 Dose response assay ISIS 0.12 0.60 3.00 15.00 No μM μM μM μM 333611 0 0 0 1 611475 0 17 0 16 611492 13 24 41 62 612928 12 36 49 72 612931 31 68 83 86 654301 2 0 0 9 654302 0 8 3 0 654309 18 7 11 9 654310 13 19 22 7 654317 3 0 1 20 654318 4 0 33 17 654325 0 0 0 14 654326 0 15 17 48 654333 19 18 36 55 654334 0 0 0 6 654341 0 9 0 25 654342 0 0 0 18 654349 0 13 31 49 654350 10 32 66 79

TABLE 44 Dose response assay ISIS 0.12 0.60 3.00 15.00 No μM μM μM μM 333611 5 0 7 3 611497 16 49 60 75 611500 9 8 21 49 612932 17 8 26 37 612941 4 12 36 51 654303 0 1 0 5 654304 9 10 27 43 654311 15 51 68 84 654312 6 26 29 33 654319 3 44 2 8 654320 4 12 5 12 654327 3 45 65 81 654328 15 44 73 85 654335 2 0 0 12 654336 0 0 0 0 654343 0 7 26 59 654344 10 30 51 72 654351 10 22 48 77 654352 8 26 57 76

Example 11: Dose-Dependent Inhibition of Human SOD-1 by Gapmers with Mixed Backbone Chemistry

Additional gapmers were designed based on the sequences of the oligonucleotides disclosed in studies described above. The oligonucleotides were designed as 5-10-5 MOE gapmers, 4-8-5 MOE gapmers, 5-8-5 MOE gapmers, 5-8-7 MOE gapmers, 6-8-6 MOE gapmers, 6-9-5 MOE gapmers, or deoxy, MOE and cEt oligonucleotides.

The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ directions comprising five nucleosides each. The 4-8-5 MOE gapmers are 17 nucleosides in length, wherein the central gap segment is comprised of eight 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ directions comprising four and five nucleosides respectively. The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ directions comprising five nucleosides each. The 5-8-7 MOE gapmers are 20 nucleosides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ directions comprising five and seven nucleosides respectively. The 6-8-6 MOE gapmers are 20 nucleosides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ directions comprising six nucleosides each. The 6-9-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ directions comprising six and five nucleosides respectively. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification.

The deoxy, MOE and cEt oligonucleotides are 17 nucleosides in length wherein each nucleoside has a MOE sugar modification, a cEt sugar modification, or a deoxy moiety. The sugar chemistry of each oligonucleotide is denoted as in the Chemistry column, where ‘k’ indicates a cEt modified sugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’ indicates a 2′-MOE modified sugar.

The internucleoside linkages throughout each gapmer are either phosphodiester or phosphorothioate linkages. The internucleoside linkages of each oligonucleotide are denoted in the Backbone Chemistry column, where ‘o’ indicates a phosphodiester linkage and ‘s’ indicates a phosphorothioate linkage. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human SOD-1 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the human SOD-1 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000 to 18704000).

TABLE 45  Modified oligonucleotides targeting human SOD-1 with mixed backbone chemistry SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: NO: 1 1 2 2 SEQ Start Stop Backbone Start Stop ID ISIS NO Site Site  Sequence Motif Sugar Modifications Chemistry Site Site NO 666846 665 684 CAGGATACAT 5-10-5 eeeeeddddddddddeeeee soooossssssssssooss 9815 9834 725 TTCTACAGCT MOE 666849 665 684 CAGGATACAT 5-10-5 eeeeeddddddddddeeeee sooosssssssssssooss 9815 9834 725 TTCTACAGCT MOE 666853 665 684 CAGGATACAT 5-10-5 eeeeeddddddddddeeeee sososssssssssssosos 9815 9834 725 TTCTACAGCT MOE 666859 679 695 TTAATGTTTA Deoxy, eeeeddddddddkkeee soosssssssssooss 9829 9845 1351 TCAGGAT MOE and cEt 666861 679 695 TTAATGTTTA Deoxy, ekekddddddddeeeee soosssssssssooss 9829 9845 1351 TCAGGAT MOE and cEt 666867 684 700 AGTGTTTAAT Deoxy, eekkddddddddeeeee soosssssssssooss 9834 9850 1173 GTTTATC MOE and cEt 666869 684 700 AGTGTTTAAT Deoxy, ekekddddddddkekee soosssssssssooss 9834 9850 1173 GTTTATC MOE and cEt 666870 684 700 AGTGTTTAAT Deoxy, ekekddddddddeeeee soosssssssssooss 9834 9850 1173 GTTTATC MOE and cEt 666919 666 682 GGATACATTT Deoxy, eeeedddddddddkkee sooosssssssssoss 9816 9832 1342 CTACAGC MOE and cEt 666921 666 682 GGATACATTT Deoxy, eeeeeddddddddkkee sooosssssssssoss 9816 9832 1342 CTACAGC MOE and cEt 666922 666 682 GGATACATTT Deoxy, eeeekddddddddkeee sooosssssssssoss 9816 9832 1342 CTACAGC MOE and cEt 684059 676 692 ATGTTTATCA Deoxy, eeekddddddddkeeee soosssssssssooss 9826 9842 1348 GGATACA MOE and cEt 684064 676 692 ATGTTTATCA Deoxy, eeeeddddddddkekee soosssssssssooss 9826 9842 1348 GGATACA MOE and cEt 684068 676 692 ATGTTTATCA 4-8-5 eeeeddddddddeeeee soosssssssssooss 9826 9842 1348 GGATACA MOE 684087 590 607 GGCGATCCCA 5-8-5 eeeeeddddddddeeeee sooosssssssssooss 9740 9757 613 ATTACACC MOE 684088 167 184 GTCGCCCTTC 5-8-5 eeeeeddddddddeeeee sooosssssssssooss 973 990 1419 AGCACGCA MOE 684095 167 186 CCGTCGCCCT 5-10-5 eeeeeddddddddddeeeee soooossssssssssooss  973 992 21 TCAGCACGCA MOE 684097 167 186 CCGTCGCCCT 5-8-7 eeeeeddddddddeeeeeee sooossssssssssoooss  973 992 21 TCAGCACGCA MOE 684101 588 607 GGCGATCCCA 6-8-6 eeeeeeddddddddeeeeee sooossssssssssoooss 9738 9757 47 ATTACACCAC MOE 684102 588 607 GGCGATCCCA 5-8-7 eeeeeddddddddeeeeeee sooossssssssssoooss 9738 9757 47 ATTACACCAC MOE 684104 588 607 GGCGATCCCA 6-9-5 eeeeeedddddddddeeeee sooo0ssssssssssooss 9738 9757 47 ATTACACCAC MOE

The newly designed oligonucleotides were tested at various doses in A431 cells. The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 5,000 cells per well and modified oligonucleotides were added to the media at 0.062 μM, 0.185 μM, 0.556 μM, 1.667 μM, 5.000 μM, and 15.000 μM concentrations of modified oligonucleotide for free uptake by the cells, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR. Human primer probe sets RTS3898 was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells.

TABLE 46 Dose response assay ISIS 0.062 0.185 0.556 1.667 5.000 15.000 IC₅₀ No μM μM μM μM μM μM (μM) 666846 18 28 45 70 69 81 0.8 666919 0 1 13 28 42 55 11.0 666849 33 29 52 62 74 82 0.6 666921 2 4 15 19 37 44 >15 666853 20 29 49 69 76 83 0.7 666922 8 7 30 33 66 59 4.1 666859 26 30 58 64 68 78 0.6 666861 6 21 44 76 68 77 1.1 666867 16 43 65 68 79 83 0.5 666869 52 68 79 88 89 91 <0.06 666870 24 37 57 77 81 86 0.4

TABLE 47 Dose response assay ISIS 0.062 0.185 0.556 1.667 5.000 15.000 IC₅₀ No μM μM μM μM μM μM (μM) 684059 7 18 38 53 68 79 1.5 684102 0 9 0 0 4 0 >15 684064 12 19 29 38 51 61 5.0 684104 0 0 0 0 0 4 >15 684068 0 4 10 33 50 56 8.0 684087 3 1 29 0 0 27 >15 684088 10 11 11 3 4 18 >15 684095 12 13 14 4 7 18 >15 684097 8 4 5 4 3 9 >15 684101 7 0 0 23 6 14 >15

The newly designed oligonucleotides were also tested at various doses in SH-SY5Y cells. The modified oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and modified oligonucleotides transfected using electroporation at 0.062 μM, 0.185 μM, 0.556 μM, 1.6676 μM, 5.000 μM, 1 and 15.000 μM concentrations of modified oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3898 was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells.

TABLE 48 Dose response assay ISIS 0.062 0.185 0.556 1.667 5.000 15.000 IC₅₀ No μM μM μM μM μM μM (μM) 666846 0 17 49 62 83 91 0.9 666919 10 3 23 35 77 78 2.2 666849 10 10 33 61 81 92 1.1 666921 0 0 12 30 56 68 4.8 666853 0 17 39 59 85 82 1.3 666922 9 0 12 33 65 76 3.2 666859 11 44 53 75 77 93 0.5 666861 0 0 34 61 81 90 1.4 666867 33 10 43 61 81 77 0.9 666869 38 49 61 83 81 84 0.2 666870 3 6 48 69 77 87 1.1

TABLE 49 Dose response assay ISIS 0.062 0.185 0.556 1.667 5.000 15.000 IC₅₀ No μM μM μM μM μM μM (μM) 684059 4 30 51 68 88 92 0.7 684102 5 2 16 25 36 61 12.0 684064 15 27 52 63 79 92 0.7 684104 0 3 20 38 61 84 2.6 684068 0 4 32 37 61 83 2.3 684087 0 3 21 31 47 66 5.8 684088 13 4 5 40 52 77 3.9 684095 16 5 19 36 68 80 2.4 684097 11 15 9 30 59 76 3.6 684101 0 0 8 23 49 66 6.6

Example 12: Inhibition of Human SOD-1 in a Transgenic Rat Model

Gapmers from the studies described above, including benchmark compound ISIS 333611, which was previously disclosed in WO 2005/040180, were tested in an SOD-1 transgenic rat model (Taconic, Cat #2148-F and 2148-M). These hemizygous rats express mutant human SOD-1 in the spinal cord.

Additional gapmers were designed based on the sequences of the oligonucleotides disclosed in studies described above. The oligonucleotides were designed as 5-9-5 MOE gapmers, 5-10-5 MOE gapmers or deoxy, MOE and cEt oligonucleotides. The 5-9-5 MOE gapmers are 19 nucleosides in length, wherein the central gap segment is comprised of nine 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ directions comprising five nucleosides each. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment is comprised of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ directions comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The deoxy, MOE and cEt oligonucleotides are 17 nucleosides in length wherein each nucleoside has a MOE sugar modification, a cEt sugar modification, or a deoxy moiety The sugar chemistry of each oligonucleotide is denoted as in the Chemistry column, where ‘k’ indicates a cEt modified sugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’ indicates a 2′-MOE modified sugar. The internucleoside linkages throughout each gapmer are either phosphodiester or phosphorothioate linkages. The internucleoside linkages of each oligonucleotide is denoted in the Backbone Chemistry column, where ‘o’ indicates a phosphodiester linkage and ‘s’ indicates a phosphorothioate linkage. All cytosine residues throughout each oligonucleotide are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Table below is targeted to either the human SOD-1 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the human SOD-1 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000 to 18704000).

TABLE 50  Modified oligonucleotides targeting human SOD-1 with mixed backbone chemistry SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: NO: 1 1 2 2 SEQ ISIS Start Stop Backbone Start Stop ID NO Site Site Sequence Motif Sugar Modifications Chemistry Site Site NO 383872 167 186 CCGTCGCCCTT 5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 973 992 21 CAGCACGCA MOE 611457 165 184 GTCGCCCTTCA 5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 971 990 54 GCACGCACA MOE 611464 164 183 TCGCCCTTCAG 5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 970 989 67 CACGCACAC MOE 611467 656 675 TTTCTACAGCT 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 9806 9825 272 AGCAGGATA MOE 611468 583 602 TCCCAATTACA 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 9733 9752 227 CCACAAGCC MOE 611472 230 249 CCCCACACCTT 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 4984 5003 145 CACTGGTCC MOE 611473 231 250 TCCCCACACCT 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 4985 5004 146 TCACTGGTC MOE 611478 644 663 GCAGGATAAC 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 9794 9813 260 AGATGAGTTA MOE 611479 645 664 AGCAGGATAA 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 9795 9814 261 CAGATGAGTT MOE 611481 655 674 TTCTACAGCTA 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 9805 9824 271 GCAGGATAA MOE 611484 660 679 TACATTTCTAC 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 9810 9829 276 AGCTAGCAG MOE 611485 661 680 ATACATTTCTA 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 9811 9830 277 CAGCTAGCA MOE 611488 124 143 GCTAGGCCAC 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 930 949 593 GCCGAGGTCC MOE 611490 402 421 GTCAGCAGTCA 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 8457 8476 666 CATTGCCCA MOE 611494 671 690 GTTTATCAGGA 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 9821 9840 728 TACATTTCT MOE 611495 673 692 ATGTTTATCAG 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 9823 9842 729 GATACATTT MOE 611498 569 588 CAAGCCAAAC 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 9719 9738 816 GACTTCCAGC MOE 611499 664 683 AGGATACATTT 5-10-5 eeeeeddddddddddeeeee  sooosssssssssssooos 9814 9833 822 CTACAGCTA MOE 612912 621 637 CTCAGACTACA Deoxy, eeekkdddddddkkeee soosssssssssooss  9771 9787 1146 TCCAAG MOE, and cEt 612915 656 672 CTACAGCTAGC Deoxy, eeekkdddddddkkeee soosssssssssooss 9806 9822 1164 AGGATA MOE, and cEt 612917 684 700 AGTGTTTAATG Deoxy, eeekkdddddddkkeee soosssssssssooss  9834 9850 1173 TTTATC MOE, and cEt 612919 621 637 CTCAGACTACA Deoxy, eekkddddddddkkeee soosssssssssooss  9771 9787 1146 TCCAAG MOE, and cEt 612923 656 672 CTACAGCTAGC Deoxy, eekkddddddddkkeee soosssssssssooss 9806 9822 1164 AGGATA MOE, and cEt 612924 674 690 GTTTATCAGGA Deoxy, eekkddddddddkkeee soosssssssssooss 9824 9840 1346 TACATT MOE, and cEt 612934 170 186 CCGTCGCCCTT Deoxy, eekkdddddddddkkee sooosssssssssoss 976 992 969 CAGCAC MOE, and cEt 612935 585 601 CCCAATTACAC Deoxy, eekkdddddddddkkee sooosssssssssoss  9735 9751 1114 CACAAG MOE, and cEt 612940 659 675 TTTCTACAGCT Deoxy, eekkdddddddddkkee sooosssssssssoss  9809 9825 1167 AGCAGG MOE, and cEt 612942 668 684 CAGGATACATT Deoxy, eekkdddddddddkkee sooosssssssssoss  9818 9834 1344 TCTACA MOE, and cEt 612943 674 690 GTTTATCAGGA Deoxy, eekkdddddddddkkee sooosssssssssoss  9824 9840 1346 TACATT MOE, and cEt 666854 665 681 AGGATACATTT 5-9-5 eeeeedddddddddeeeee  sooossssssssssooss 9815 9831 1428 CTACAGCT MOE 666855 666 682 CAGGATACATT 5-9-5 eeeeedddddddddeeeee  sooossssssssssooss 9816 9832 1461 TCTACAGC MOE 666857 679 695 TTAATGTTTAT Deoxy, eeekddddddddkeeee soosssssssssooss  9829 9845 1351 CAGGAT MOE, and cEt 666858 679 695 TTAATGTTTAT Deoxy, eekkddddddddeeeee soosssssssssooss  9829 9845 1351 CAGGAT MOE, and cEt 666864 679 695 TTAATGTTTAT Deoxy, kekeddddddddeeeee soosssssssssooss  9829 9845 1351 CAGGAT MOE, and cEt 666865 679 695 TTAATGTTTAT Deoxy, eeeeddddddddekeke soosssssssssooss 9829 9845 1351 CAGGAT MOE, and cEt 666866 684 700 AGTGTTTAATG Deoxy, eeekddddddddkeeee soosssssssssooss 9834 9850 1173 TTTATC MOE, and cEt 666908 686 702 ACAGTGTTTAA Deoxy, eeeekdddddddkeeee sooossssssssooss 9836 9852 1175 TGTTTA MOE, and cEt 666923 666 682 GGATACATTTC Deoxy, eeekddddddddkeeee sooossssssssooss 9816 9832 1342 TACAGC MOE, and cEt

The modified oligonucleotides were tested in a series of experiments that had similar conditions. The results for each experiment are presented in separate tables shown below. Rats were injected intrathecally with 30 μL of a 16.67 mg/ml solution of modified oligonucleotide diluted in PBS (500 μg final dose). A control group of rats was injected intrathecally with PBS. Inhibition levels of SOD-1 in the lumbar spinal cord, thoracic spinal cord and cervical spinal cord were assessed. The data is presented below and indicate that several modified oligonucleotides inhibited human SOD-1 levels in this model.

TABLE 51 Percent inhibition of human SOD-1 in the spinal cord regions of transgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO 333611 5-10-5 MOE with 51 51 47 21 phosphorothioate backbone chemistry 383872 5-10-5 MOE 29 36 26 21 with mixed backbone chemistry 611460 5-10-5 MOE 55 53 25 1428 with mixed backbone chemistry 611464 5-10-5 MOE 52 54 26 67 with mixed backbone chemistry 611468 5-10-5 MOE 46 44 19 227 with mixed backbone chemistry 611481 5-10-5 MOE 39 44 33 271 with mixed backbone chemistry

TABLE 52 Percent inhibition of human SOD-1 in the spinal cord regions of transgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO 611473 5-10-5 MOE 47 42 5 146 with mixed backbone chemistry 611474 5-10-5 MOE 75 65 65 149 with mixed backbone chemistry 611479 5-10-5 MOE 24 13 20 261 with mixed backbone chemistry 611484 5-10-5 MOE 51 31 41 276 with mixed backbone chemistry 611485 5-10-5 MOE 52 40 35 277 with mixed backbone chemistry 611492 5-10-5 MOE 57 44 43 725 with mixed backbone chemistry

TABLE 53 Percent inhibition of human SOD-1 in the spinal cord regions of transgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO 611472 5-10-5 MOE 0 19 15 145 with mixed backbone chemistry 611478 5-10-5 MOE 16 33 24 260 with mixed backbone chemistry 611490 5-10-5 MOE 53 55 44 666 with mixed backbone chemistry 611494 5-10-5 MOE 34 39 38 728 with mixed backbone chemistry 611495 5-10-5 MOE 33 19 38 729 with mixed backbone chemistry 611498 5-10-5 MOE 30 43 27 816 with mixed backbone chemistry 611499 5-10-5 MOE 45 56 40 822 with mixed backbone chemistry 611500 5-10-5 MOE 56 58 52 823 with mixed backbone chemistry

TABLE 54 Percent inhibition of human SOD-1 in the spinal cord regions of transgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO 611457 5-10-5 MOE 56 46 43 54 with mixed backbone chemistry 611467 5-10-5 MOE 21 28 22 272 with mixed backbone chemistry 611488 5-10-5 MOE 14 23 4 593 with mixed backbone chemistry 612917 Deoxy, MOE, and cEt 47 55 37 1173 with mixed backbone chemistry 612923 Deoxy, MOE, and cEt 53 63 45 1164 with mixed backbone chemistry 612925 Deoxy, MOE, and cEt 67 69 63 1348 with mixed backbone chemistry 612928 Deoxy, MOE, and cEt 84 85 81 1351 with mixed backbone chemistry

TABLE 55 Percent inhibition of human SOD-1 in the spinal cord regions of transgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO 612912 Deoxy, MOE, and cEt 59 60 48 1146 with mixed backbone chemistry 612919 Deoxy, MOE, and cEt 60 60 58 1146 with mixed backbone chemistry 612916 Deoxy, MOE, and cEt 72 69 69 1170 with mixed backbone chemistry 612931 Deoxy, MOE, and cEt 81 79 72 1173 with mixed backbone chemistry 612932 Deoxy, MOE, and cEt 21 26 24 1175 with mixed backbone chemistry

TABLE 56 Percent inhibition of human SOD-1 in the spinal cord regions of transgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO 612915 Deoxy, MOE, and cEt 54 48 52 1164 with mixed backbone chemistry 612918 Deoxy, MOE, and cEt 73 69 64 1175 with mixed backbone chemistry 612927 Deoxy, MOE, and cEt 82 75 62 1350 with mixed backbone chemistry 612934 Deoxy, MOE, and cEt 59 44 48 969 with mixed backbone chemistry 612935 Deoxy, MOE, and cEt 64 54 62 1114 with mixed backbone chemistry 612940 Deoxy, MOE, and cEt 11 26 17 1167 with mixed backbone chemistry 612941 Deoxy, MOE, and cEt 81 75 71 1342 with mixed backbone chemistry 612942 Deoxy, MOE, and cEt 40 42 41 1344 with mixed backbone chemistry 612943 Deoxy, MOE, and cEt 61 54 51 1346 with mixed backbone chemistry 612944 Deoxy, MOE, and cEt 59 52 51 1348 with mixed backbone chemistry

TABLE 57 Percent inhibition of human SOD-1 in the spinal cord regions of transgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO 612924 Deoxy, MOE, and cEt 42 64 53 1346 with mixed backbone chemistry 612947 Deoxy, MOE, and cEt 68 75 74 1351 with mixed backbone chemistry 612948 Deoxy, MOE, and cEt 80 90 87 1352 with mixed backbone chemistry 612949 Deoxy, MOE, and cEt 73 82 85 1172 with mixed backbone chemistry

TABLE 58 Percent inhibition of human SOD-1 in the spinal cord regions of transgenic rats ISIS SEQ No Chemistry Lumbar Cervical ID NO 654304 5-8-5 MOE 28 6 1429 with mixed backbone chemistry 654305 5-8-5 MOE 14 0 1430 with mixed backbone chemistry 654306 5-8-5 MOE 36 0 1432 with mixed backbone chemistry 654307 5-8-5 MOE 17 0 1432 with mixed backbone chemistry

TABLE 59 Percent inhibition of human SOD-1 in the spinal cord regions of transgenic rats ISIS SEQ No Chemistry Lumbar Cervical ID NO 654334 Deoxy, MOE, and cEt 39 19 1454 with mixed backbone chemistry 666854 5-9-5 MOE 52 39 1428 with mixed backbone chemistry 666855 5-9-5 MOE 37 17 1461 with mixed backbone chemistry 666857 Deoxy, MOE, and cEt 59 39 1351 with mixed backbone chemistry 666858 Deoxy, MOE, and cEt 38 22 1351 with mixed backbone chemistry 666859 Deoxy, MOE, and cEt 79 64 1351 with mixed backbone chemistry 666864 Deoxy, MOE, and cEt 50 40 1351 with mixed backbone chemistry 666865 Deoxy, MOE, and cEt 73 44 1351 with mixed backbone chemistry 666866 Deoxy, MOE, and cEt 67 56 1173 with mixed backbone chemistry 666908 Deoxy, MOE, and cEt 38 13 1175 with mixed backbone chemistry 666923 Deoxy, MOE, and cEt 45 26 1342 with mixed backbone chemistry

TABLE 60 Percent inhibition of human SOD-1 in the spinal cord regions of transgenic rats ISIS SEQ No Chemistry Lumbar Cervical ID NO 654323 Deoxy, MOE, and cEt 53 35 1433 with mixed backbone chemistry 666846 5-10-5 MOE 64 50 725 with mixed backbone chemistry 666849 5-10-5 MOE 63 55 725 with mixed backbone chemistry 666853 5-10-5 MOE 81 74 725 with mixed backbone chemistry 666861 Deoxy, MOE, and cEt 55 47 1351 with mixed backbone chemistry 666867 Deoxy, MOE, and cEt 59 48 1173 with mixed backbone chemistry 666869 Deoxy, MOE, and cEt 82 81 1173 with mixed backbone chemistry 666870 Deoxy, MOE, and cEt 76 68 1173 with mixed backbone chemistry 666919 Deoxy, MOE, and cEt 76 68 1342 with mixed backbone chemistry 666921 Deoxy, MOE, and cEt 71 65 1342 with mixed backbone chemistry 666922 Deoxy, MOE, and cEt 67 62 1342 with mixed backbone chemistry

TABLE 61 Percent inhibition of human SOD-1 in the spinal cord regions of transgenic rats ISIS SEQ ID No Chemistry Lumbar NO 684059 Deoxy, MOE, and cEt 54 1348 with mixed backbone chemistry 684064 Deoxy, MOE, and cEt 51 1348 with mixed backbone chemistry 684068 4-8-5 MOE 18 1348 with mixed backbone chemistry 684087 5-8-5 MOE 37 613 with mixed backbone chemistry 684088 5-8-5 MOE 31 1419 with mixed backbone chemistry 684095 5-10-5 MOE 34 21 with mixed backbone chemistry 684097 5-8-7 MOE 22 21 with mixed backbone chemistry 684101 6-8-6 MOE 22 47 with mixed backbone chemistry 684104 6-9-5 MOE 11 47 with mixed backbone chemistry

Example 13: Dose-Dependent Inhibition of Human SOD-1 with Modified Oligonucleotides in LLC-MK2 Cells

Gapmers from the studies described above, including benchmark compound ISIS 333611, exhibiting significant in vitro inhibition of SOD-1 mRNA were selected and tested at various doses in LLC-MK2 cells. The cross-reactivity of the human modified oligonucleotides tested in this study with the rhesus monkey genomic sequence (the complement of GENBANK Accession No. NW_001114168.1 truncated from nucleotides 2258000 to 2271000, designated herein as SEQ ID NO: 3) is shown in the Table below.

TABLE 62 Cross-reactivity of antisense oligonucleotides targeting human SOD1with SEQ ID NO: 3 ISIS Start Site of No SEQ ID NO: 3 Mismatches 333611 1572 2 436839 1564 0 436854 9049 0 436867 10347 0 666853 10375 1 666859 10389 1 666919 10376 1 666921 10376 1

Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.078 μM, 0.156 μM, 0.313 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM, and 10.000 μM concentrations of modified oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and SOD-1 mRNA levels were measured by quantitative real-time PCR Primer probe set RTS3121 (forward sequence TGGAGATAATACACAAGGCTGTACCA, designated herein as SEQ ID NO: 17; reverse sequence CAACATGCCTCTCTTCATCCTTT, designated herein as SEQ ID NO: 18; probe sequence ATCCTCTATCCAGACAACACGGTGGGC, designated herein as SEQ ID NO: 19) was used to measure mRNA levels. SOD-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of SOD-1, relative to untreated control cells. The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. As presented in the Table, several of the newly designed oligonucleotides were more potent than the benchmark, ISIS 336611.

TABLE 63 Dose-dependent inhibition of SOD-1 rhesus monkey mRNA ISIS 0.078 0.156 0.313 0.625 1.25 2.50 5.00 10.00 IC₅₀ No μM μM μM μM μM μM μM μM (μM) 333611 3 2 0 0 17 15 19 40 >10 436839 0 0 5 0 20 22 37 61 7.1 436854 34 34 40 39 52 65 69 84 1.2 436867 3 0 11 18 34 49 70 87 2.2 666853 7 34 20 39 60 80 79 92 1.0 666859 0 9 20 18 15 25 30 44 >10 666919 11 15 16 36 51 65 73 84 1.4 666921 0 13 28 37 50 52 62 74 1.8

Example 14: Tolerability of SOD-1 Modified Oligonucleotides in a Rat Model

Gapmers from the studies described above, including benchmark compound ISIS 333611, which was previously disclosed in WO 2005/040180, were tested for tolerability in Sprague-Dawley rats.

The modified oligonucleotides were tested in a series of experiments that had similar conditions. Rats were injected intrathecally with 3 mg of a single dose of ISIS oligonucleotide. A control group of rats was injected intrathecally with PBS. Acute tolerability was assessed 3 hours post-dose using a functional observational battery (FOB). This score is used to evaluate the acute tolerability of a compound with lower scores denoting better tolerated compounds. Control animals usually have a score of ‘0’ or ‘1’. At 3 hours post injection, the rats are observed by placing each rat on the cage top and evaluating certain functions, assigning a number of ‘0’ or ‘1’ depending on whether the rat exhibits normal function in the region of interest (0) or does not (1) for each function, and then adding the total scores. Seven regions are assessed, including tail, hind paws, hind legs, hind end, front posture, fore paws, and head. The results of the scoring are presented in the Table below. As presented in the Table, several newly designed oligonucleotides demonstrated more acute tolerability compared to the benchmark, ISIS 333611.

TABLE 64 FOB scores in Sprague-Dawley rats Target Start ISIS Site on SEQ FOB No ID NO: 1 Chemistry score 333611 167 5-10-5 MOE with 4 phosphorothioate backbone 684073 167 Deoxy, MOE, and cEt with 3 mixed backbone 684081 167 Deoxy, MOE, and cEt with 1 mixed backbone 684088 167 5-8-5 MOE with mixed 0 backbone 684093 167 5-9-5 MOE with mixed 0 backbone 684095 167 5-10-5 MOE with mixed 0 backbone 684096 167 6-8-6 MOE with mixed 0 backbone 684097 167 5-8-7 MOE with mixed 0 backbone 684098 167 7-8-5 MOE with mixed 0 backbone 684099 167 6-9-5 MOE with mixed 0 backbone 684074 168 Deoxy, MOE, and cEt with 0 mixed backbone 684082 168 Deoxy, MOE, and cEt with 1 mixed backbone 684089 168 5-8-5 MOE with mixed 0 backbone 684094 168 5-9-5 MOE with mixed 1 backbone 684075 169 Deoxy, MOE, and cEt with 3 mixed backbone 684083 169 Deoxy, MOE, and cEt with 3 mixed backbone 684090 169 5-8-5 MOE with mixed 2 backbone 684076 170 Deoxy, MOE, and cEt with 2 mixed backbone 684084 170 Deoxy, MOE, and cEt with 4 mixed backbone 611474 234 5-10-5 MOE with mixed 4 backbone 654301 234 5-8-5 MOE with mixed 3 backbone 654302 235 5-8-5 MOE with mixed 1 backbone 654303 236 5-8-5 MOE with mixed 0 backbone 684069 588 Deoxy, MOE, and cEt with 0 mixed backbone 684077 588 Deoxy, MOE, and cEt with 1 mixed backbone 684085 588 5-8-5 MOE with mixed 0 backbone 684091 588 5-9-5 MOE with mixed 0 backbone 684100 588 5-10-5 MOE with mixed 0 backbone 684101 588 6-8-6 MOE with mixed 0 backbone 684102 588 5-8-7 MOE with mixed 0 backbone 684103 588 7-8-5 MOE with mixed 0 backbone 684104 588 6-9-5 MOE with mixed 0 backbone 684070 589 Deoxy, MOE, and cEt with 0 mixed backbone 684078 589 Deoxy, MOE, and cEt with 0 mixed backbone 684086 589 5-8-5 MOE with mixed 0 backbone 684092 589 5-9-5 MOE with mixed 0 backbone 684071 590 Deoxy, MOE, and cEt with 0 mixed backbone 684079 590 Deoxy, MOE, and cEt with 0 mixed backbone 684087 590 5-8-5 MOE with mixed 0 backbone 684072 591 Deoxy, MOE, and cEt with 1 mixed backbone 684080 591 Deoxy, MOE, and cEt with 1 mixed backbone 654304 663 5-8-5 MOE with mixed 3 backbone 612916 664 Deoxy, MOE, and cEt with 0 mixed backbone 654305 664 5-8-5 MOE with mixed 2 backbone 611492 665 5-10-5 MOE with mixed 0 backbone 654306 665 5-8-5 MOE with mixed 3 backbone 654323 665 Deoxy, MOE, and cEt with 0 mixed backbone 654341 665 Deoxy, MOE, and cEt with 0 mixed backbone 666846 665 5-10-5 MOE with mixed 0 backbone 666849 665 5-10-5 MOE with mixed 0 backbone 666851 665 5-10-5 MOE with mixed 1 backbone 666853 665 5-10-5 MOE with mixed 0 backbone 666854 665 5-9-5 MOE with mixed 1 backbone 611500 666 5-10-5 MOE with mixed 0 backbone 612941 666 Deoxy, MOE, and cEt with 3 mixed backbone 654307 666 5-8-5 MOE with mixed 2 backbone 654342 666 Deoxy, MOE, and cEt with 2 mixed backbone 666845 666 5-10-5 MOE with mixed 0 backbone 666848 666 5-10-5 MOE with mixed 1 backbone 666850 666 5-10-5 MOE with mixed 0 backbone 666852 666 5-10-5 MOE with mixed 1 backbone 666855 666 5-9-5 MOE with mixed 1 backbone 666917 666 Deoxy, MOE, and cEt with 3 mixed backbone 666918 666 Deoxy, MOE, and cEt with 3 mixed backbone 666919 666 Deoxy, MOE, and cEt with 2 mixed backbone 666920 666 Deoxy, MOE, and cEt with 1 mixed backbone 666921 666 Deoxy, MOE, and cEt with 2 mixed backbone 666922 666 Deoxy, MOE, and cEt with 3 mixed backbone 666923 666 Deoxy, MOE, and cEt with 2 mixed backbone 666856 667 5-9-5 MOE with mixed 3 backbone 612925 676 Deoxy, MOE, and cEt with 4 mixed backbone 684059 676 Deoxy, MOE, and cEt with 4 mixed backbone 684060 676 Deoxy, MOE, and cEt with 3 mixed backbone 684061 676 Deoxy, MOE, and cEt with 4 mixed backbone 684062 676 Deoxy, MOE, and cEt with 4 mixed backbone 684063 676 Deoxy, MOE, and cEt with 5 mixed backbone 684064 676 Deoxy, MOE, and cEt with 4 mixed backbone 684065 676 Deoxy, MOE, and cEt with 4 mixed backbone 684066 676 Deoxy, MOE, and cEt with 4 mixed backbone 684067 676 Deoxy, MOE, and cEt with 5 mixed backbone 684068 676 4-8-5 MOE with mixed 4 backbone 612927 678 Deoxy, MOE, and cEt with 4 mixed backbone 654309 678 5-8-5 MOE with mixed 4 backbone 612928 679 Deoxy, MOE, and cEt with 2 mixed backbone 612947 679 Deoxy, MOE, and cEt with 7 mixed backbone 654310 679 5-8-5 MOE with mixed 3 backbone 666857 679 Deoxy, MOE, and cEt with 1 mixed backbone 666858 679 Deoxy, MOE, and cEt with 1 mixed backbone 666859 679 Deoxy, MOE, and cEt with 1 mixed backbone 666860 679 Deoxy, MOE, and cEt with 0 mixed backbone 666861 679 Deoxy, MOE, and cEt with 5 mixed backbone 666862 679 Deoxy, MOE, and cEt with 1 mixed backbone 666863 679 Deoxy, MOE, and cEt with 4 mixed backbone 666864 679 Deoxy, MOE, and cEt with 4 mixed backbone 666865 679 Deoxy, MOE, and cEt with 5 mixed backbone 611497 681 5-10-5 MOE with mixed 5 backbone 612948 681 Deoxy, MOE, and cEt with 3 mixed backbone 666847 681 5-10-5 MOE with mixed 7 backbone 612949 683 Deoxy, MOE, and cEt with 4 mixed backbone 612931 684 Deoxy, MOE, and cEt with 4 mixed backbone 666866 684 Deoxy, MOE, and cEt with 6 mixed backbone 666867 684 Deoxy, MOE, and cEt with 6 mixed backbone 666868 684 Deoxy, MOE, and cEt with 6 mixed backbone 666869 684 Deoxy, MOE, and cEt with 6 mixed backbone 666870 684 Deoxy, MOE, and cEt with 6 mixed backbone 666871 684 Deoxy, MOE, and cEt with 6 mixed backbone 666872 684 Deoxy, MOE, and cEt with 6 mixed backbone 666873 684 Deoxy, MOE, and cEt with 6 mixed backbone 666874 684 Deoxy, MOE, and cEt with 5 mixed backbone 612918 686 Deoxy, MOE, and cEt with 4 mixed backbone 612932 686 Deoxy, MOE, and cEt with 2 mixed backbone 666906 686 Deoxy, MOE, and cEt with 2 mixed backbone 666907 686 Deoxy, MOE, and cEt with 3 mixed backbone 666908 686 Deoxy, MOE, and cEt with 1 mixed backbone 666909 686 Deoxy, MOE, and cEt with 0 mixed backbone 666910 686 Deoxy, MOE, and cEt with 2 mixed backbone 666911 686 Deoxy, MOE, and cEt with 0 mixed backbone 666912 686 Deoxy, MOE, and cEt with 0 mixed backbone 666913 686 Deoxy, MOE, and cEt with 0 mixed backbone 666914 686 Deoxy, MOE, and cEt with 0 mixed backbone 666915 686 Deoxy, MOE, and cEt with 1 mixed backbone 666916 686 Deoxy, MOE, and cEt with 1 mixed backbone 654318 687 5-8-5 MOE with mixed 1 backbone 654334 687 Deoxy, MOE, and cEt with 3 mixed backbone

Tolerability was also assessed 8 weeks post-dose by measuring the levels of IBA1, a microglial marker, and GFAP, an astrocytic marker, in the lumbar spinal cord region. Both IBA1 and GFAP are markers of CNS inflammation (Frank, M G, Brain Behav. Immun. 2007, 21, 47-59), hence the higher the level of either marker, the less tolerable the antisense oligonucleotide is deemed to be in this rat model.

IBA1 mRNA levels were measured with primer probe set rAIF1_LTS00219 (forward sequence AGGAGAAAAACAAAGAACACCAGAA, designated herein as SEQ ID NO: 5; reverse sequence CAATTAGGGCAACTCAGAAATAGCT, designated herein as SEQ ID NO: 6; probe sequence CCAACTGGTCCCCCAGCCAAGA, designated herein as SEQ ID NO: 7). GFAP mRNA levels were measured with primer probe set mGFAP_LTS00370 (forward sequence GAAACCAGCCTGGACACCAA, designated herein as SEQ ID NO: 8; reverse sequence TCCACAGTCTTTACCACGATGTTC, designated herein as SEQ ID NO: 9; probe sequence TCCGTGTCAGAAGGCCACCTCAAGA, designated herein as SEQ ID NO: 10).

The results are presented in the Table below. As presented in the Table, several newly designed oligonucleotides were more tolerable compared to the benchmark, ISIS 333611.

TABLE 65 IBA1 and GFAP mRNA levels (% control) in the lumbar regions of Sprague-Dawley rats ISIS No. IBA1 GFAP 333611 341 314 654301 149 137 654302 261 129 654303 110 80 654304 143 130 654305 185 158 654306 110 106 654307 152 144 654309 195 169 654310 119 141 654318 93 81 654323 125 113 654334 114 75 654341 209 224 654342 473 485 666845 389 416 666846 173 171 666847 271 297 666848 399 377 666849 140 150 666850 246 252 666851 246 199 666852 282 266 666853 168 147 666854 135 123 666855 238 221 666856 253 209 666857 242 182 666858 169 134 666859 185 162 666861 161 152 666862 254 285 666863 216 185 666864 174 154 666865 251 232 666866 281 135 666867 132 112 666868 199 211 666869 262 207 666870 201 189 666871 192 214 666872 441 136 666873 340 277 666874 204 199 666917 292 244 666919 115 85 666920 155 102 666921 108 82 666922 123 82 666923 118 93 684059 168 162 684060 158 141 684061 335 263 684062 218 265 684064 191 168 684065 245 304 684066 313 376 684067 171 151 684068 157 135 684085 459 586 684086 187 227 684087 215 263 684088 151 183 684089 507 667 684090 130 170 684091 350 426 684092 366 333 684093 412 264 684094 294 373 684095 213 215 684096 404 335 684097 217 206 684098 378 438 684099 534 473 684100 276 259 684101 153 125 684102 237 242 684103 588 416 684104 221 193

Example 15: Dose Dependent Inhibition of Human SOD-1 in a Transgenic Rat Model

Gapmers from the studies described above, including benchmark compound ISIS 333611, were tested in an SOD-1 transgenic rat model (Taconic, Cat #2148-F and 2148-M). These hemizygous rats express mutant human SOD-1 in the spinal cord, many brain regions, and peripheral organs. Rats were injected intrathecally with 10, 30, 100, 300, 1000, or 3000 μg of a gapmer listed in the table below or with only PBS. Two weeks later, the animals were sacrificed. Inhibition of SOD-1 mRNA in the lumbar spinal cord, cervical spinal cord, rostral cortex, and caudal cortex was assessed by RT-PCR using primer probe set RTS3898, described in Example 1. The data is presented below as ED₅₀ values, and indicates that the oligonucleotides inhibited SOD1 mRNA in multiple CNS tissues more potently than Isis 333611. Indeed, ED₅₀ values for Isis No. 333611 could not even be calculated, as indicated by an entry of “n/a,” because even the highest concentration tested (3000 μg) did not inhibit SOD-1 mRNA greater than 55-65%. “n.d.” indicates that there is no data available for the indicated sample.

TABLE 66 Inhibition of human SOD1 in transgenic rats ED₅₀ (μg) Isis No. Lumbar Cervical Rostral Caudal SEQ ID NO. 333611 n/a n/a n.d. n.d. 21 666853 81.3 242.6 6434 931 725 666859 74.0 358.8 2360 1113 1351 666870 139.4 1111 5511 2105 1173 666919 104.1 613.5 >6000 2655 1342

Example 16: Tolerability of SOD-1 Modified Oligonucleotides in Rats

Gapmers from the studies described above, including benchmark compound ISIS 333611, were tested for tolerability in Sprague-Dawley rats. Groups of 4 to 6 rats were injected intrathecally with 1 mg or 3 mg of a single dose of an ISIS oligonucleotide. A control group of rats was injected intrathecally with PBS. Acute tolerability was assessed 3 hours post-dose, as described in Example 14. The results for the 1 mg dose are the averages for each group following one experiment. The results for the 3 mg dose are the averages for each group across two replicate experiments. The results of the study, presented in the table below, indicate that several newly designed oligonucleotides were more tolerable than the benchmark, ISIS 333611.

TABLE 67 FOB values Isis 3 hour FOB 8 week FOB No. 1 mg 3 mg 1 mg 3 mg 333611 3.0 4.9 0.0 1.2 666853 0.0 0.5 0.0 0.0 666859 0.0 2.1 0.0 0.3 666870 2.3 5.8 0.0 0.8 666919 1.3 3.5 0.0 0.1

Example 17: Dose Dependent Inhibition of Human SOD-1 in a Transgenic Mouse Model

In order to confirm the results obtained in transgenic rats in another species, gapmers from the studies described above were tested in an SOD-1 transgenic mouse model that expresses the same G93A human mutant SOD1 gene that the transgenic rat expresses (see Examples 12 and 15).

Mice received an intracerebral ventricular bolus (ICVB) of 10, 30, 100, 300, or 700 μg of a gapmer listed in the table below, or PBS. Two weeks later, the animals were sacrificed. Inhibition of SOD-1 mRNA in the lumbar spinal cord and cortex was assessed by RT-PCR using primer probe set RTS3898, described in Example 1. The data is presented below as ED₅₀ values, and indicates that the oligonucleotides inhibited SOD1 mRNA more potently than Isis 333611 in both rats and mice.

TABLE 68 Inhibition of human SOD1 in transgenic mice Isis No. Lumbar ED₅₀ (μg) Cortex ED₅₀ (μg) 333611 401 786 666853 136 188 666859 106 206 666870 148 409 666919 168 1211

Example 18: Tolerability of SOD-1 Modified Oligonucleotides in Mice

Gapmers from the studies described above, including benchmark compound ISIS 333611, were tested for tolerability in C57bl6 mice. Mice were injected stereotaxically into the cerebral ventricles with 700 ug of a single dose of ISIS oligonucleotide. A control group of mice was injected into the cerebral ventricle with PBS. Acute tolerability was assessed at 3 hours post injection using a functional observation battery (FOB) different from that used for the rats. Each mouse was evaluated according to 7 different criteria. The 7 criteria are (1) the mouse was bright, alert, and responsive; (2) the mouse was standing or hunched without stimuli; (3) the mouse shows any movement without stimuli (4) the mouse demonstrates forward movement after its lifted; (5) the mouse demonstrates any movement after its lifted; (6) the mouse responds to a tail pinch; (7) regular breathing. For each of the 7 different criteria, each mouse was given a sub-score of 0 if it met the criteria or 1 if it did not. After all of the 7 criteria were evaluated, the sub-scores were summed for each mouse and then averaged for each group. For example, if a mouse was bright, alert, and responsive 3 hours after the 700 μg ICV dose, and met all other other criteria, it would get a summed score of 0. If another mouse was not bright, alert, and responsive 3 hours after the 700 μg ICV dose but met all other criteria, it would receive a score of 1. Saline treated mice generally receive a score of 0. A score at the top end of the range would be suggestive of acute toxicity.

Bodyweights were measured throughout the study and are reported below as percent change at 8 weeks relative to baseline. Long term tolerability was assessed 8 weeks post-dose by measuring the levels of IBA1 and GFAP, as described in Example 14. IBA1 and GFAP mRNA levels are reported relative to PBS treated animals. The results of the study, presented in the tables below, indicate that several newly designed oligonucleotides were more tolerable, in rats and mice, compared to the benchmark, ISIS 333611.

TABLE 69 FOB values and body weight change Isis No. 3 hour FOB Body weight (% change) 333611 6.5 3.8 666853 1.25 8.0 666859 1.75 14.0 666870 4.75 7.3 666919 0.0 5.2

TABLE 70 Inflammation markers Isis IBA1 (% PBS) GFAP (% PBS) No. Lumbar Cortex Lumbar Cortex 333611 130.3 134.3 117.5 207.7 666853 102.8 109.3 103.3 103.7 666859 110.4 98.2 109.0 72.8 666870 158.8 117.8 106.7 128.6 666919 115.0 97.9 99.8 84.3

Example 19: Dose Dependent Inhibition of Monkey SOD-1 in Cynomolgus Monkey

Isis No. 666853 was tested in cynomolgus monkey. There is one mismatch between Isis No. 666853 and cynomolgus monkey SOD-1, and there are 17 contiguous bases in Isis No. 666853 that are 100% complementary to cynomolgus monkey SOD-1.

Groups of 6-10 male and female monkeys received an intrathecal lumbar bolus of PBS or 4, 12, or 35 mg of Isis No. 666853 on days 1, 14, 28, 56, and 84 of the study. Each group received the same dose on all five dosing days. On day 91, the animals were sacrificed. Inhibition of SOD-1 mRNA in the lumbar, thoracic, and cervical spinal cord and frontal cortex, motor cortex, hippocampus, pons, and cerebellum was assessed by RT-PCR using primer probe set RTS3898. The data is presented below as the average percent inhibition for each treatment group, relative to the PBS treated group. The results indicate that Isis No. 666853 inhibited SOD-1 mRNA in multiple target tissues in cynomolgus monkey.

Treatment with 666853 was well tolerated for the duration of the 13 week study and there were no clinical observations of adverse reactions in monkeys.

TABLE 71 Inhibition of SOD-1 mRNA in monkeys Amount Inhibition (%) of 666853 Frontal Motor per dose (mg) Lumbar Thoracic Cervical cortex cortex Hippocampus Pons Cerebellum 4 44.4 27.1 20.1 21.5 21.6 32.0 6.8 15.4 12 75.4 69.0 42.1 56.7 55.7 31.8 13.2 33.3 35 87.0 74.8 72.1 80.5 82.6 80.1 48.6 48.4 

What is claimed is:
 1. A method for treating a superoxide dismutase 1 (SOD1) associated neurodegenerative disorder in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent; and an antisense oligonucleotide having the following formula: 5′-mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds Tds Tds mCds Tds Ads mCeo Aes Geo mCes Te -3′ (nucleobase sequence of SEQ ID NO: 725); wherein, A=an adenine, mC=a 5-methylcytosine, G=a guanine, T=a thymine, e=a 2′-O-methoxyethylribose modified sugar, d=a 2′-deoxyribose sugar, s=a phosphorothioate internucleoside linkage, and o=a phosphodiester internucleoside linkage; or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein the pharmaceutically acceptable carrier or diluent is a sterile aqueous solution.
 3. The method of claim 1, wherein the pharmaceutically acceptable carrier or diluent is phosphate buffered saline (PBS).
 4. The method of claim 1, wherein the pharmaceutical composition is administered intrathecally.
 5. The method of claim 1, wherein the SOD1 associated neurodegenerative disorder is amyotrophic lateral sclerosis (ALS) associated with a mutation in the SOD1 gene.
 6. The method of claim 5, wherein the mutation is a missense mutation.
 7. The method of claim 5, wherein the mutation is a gain of function mutation.
 8. The method of claim 5, wherein the ALS is familial SOD1 associated ALS.
 9. The method of claim 5, wherein the ALS is sporadic SOD1 associated ALS.
 10. The method of claim 5, wherein the pharmaceutical composition is administered intrathecally.
 11. The method of claim 8, wherein the pharmaceutical composition is administered intrathecally.
 12. The method of claim 9, wherein the pharmaceutical composition is administered intrathecally.
 13. A method for treating a superoxide dismutase 1 (SOD1) associated neurodegenerative disorder in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent; and an antisense oligonucleotide according to the following formula:

or a pharmaceutically acceptable salt thereof.
 14. The method of claim 13, wherein the pharmaceutically acceptable carrier or diluent is a sterile aqueous solution.
 15. The method of claim 13, wherein the pharmaceutically acceptable carrier or diluent is PBS.
 16. The method of claim 13, wherein the pharmaceutical composition is administered intrathecally.
 17. The method of claim 13, wherein the SOD1 associated neurodegenerative disorder is amyotrophic lateral sclerosis (ALS) associated with a mutation in the SOD1 gene.
 18. The method of claim 17, wherein the mutation is a missense mutation.
 19. The method of claim 17, wherein the mutation is a gain of function mutation.
 20. The method of claim 17, wherein the ALS is familial SOD1 associated ALS.
 21. The method of claim 17, wherein the ALS is sporadic SOD1 associated ALS.
 22. The method of claim 17, wherein the pharmaceutical composition is administered intrathecally.
 23. The method of claim 20, wherein the pharmaceutical composition is administered intrathecally.
 24. The method of claim 21, wherein the pharmaceutical composition is administered intrathecally. 